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Sample records for itraq proteomic identification

  1. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics.

    Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

    2016-04-29

    Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

    Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

    2015-05-01

    The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Identification of novel biomarkers for sepsis prognosis via urinary proteomic analysis using iTRAQ labeling and 2D-LC-MS/MS.

    Longxiang Su

    Full Text Available Sepsis is the major cause of death for critically ill patients. Recent progress in proteomics permits a thorough characterization of the mechanisms associated with critical illness. The purpose of this study was to screen potential biomarkers for early prognostic assessment of patients with sepsis.For the discovery stage, 30 sepsis patients with different prognoses were selected. Urinary proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ coupled with LC-MS/MS. Mass spec instrument analysis were performed with Mascot software and the International Protein Index (IPI; bioinformatic analyses were used by the algorithm of set and the Gene Ontology (GO Database. For the verification stage, the study involved another 54 sepsis-hospitalized patients, with equal numbers of patients in survivor and non-survivor groups based on 28-day survival. Differentially expressed proteins were verified by Western Blot.A total of 232 unique proteins were identified. Proteins that were differentially expressed were further analyzed based on the pathophysiology of sepsis and biomathematics. For sepsis prognosis, five proteins were significantly up-regulated: selenium binding protein-1, heparan sulfate proteoglycan-2, alpha-1-B glycoprotein, haptoglobin, and lipocalin; two proteins were significantly down-regulated: lysosome-associated membrane proteins-1 and dipeptidyl peptidase-4. Based on gene ontology clustering, these proteins were associated with the biological processes of lipid homeostasis, cartilage development, iron ion transport, and certain metabolic processes. Urinary LAMP-1 was down-regulated, consistent with the Western Blot validation.This study provides the proteomic analysis of urine to identify prognostic biomarkers of sepsis. The seven identified proteins provide insight into the mechanism of sepsis. Low urinary LAMP-1 levels may be useful for early prognostic assessment of sepsis.ClinicalTrial.gov NCT01493492.

  4. Data from proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis

    Yongxin Yang

    2015-06-01

    Full Text Available Milk fat globules memebrane (MFGM-enriched proteomes from Holstein, Jersey, yak, buffalo, goat, camel, horse, and human were extracted and identified by an iTRAQ quantification proteomic approach. Proteomes data were analyzed by bioinformatic and multivariate statistical analysis and used to present the characteristic traits of the MFGM proteins among the studied mammals. The data of this study are also related to the research article “Proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis” in the Journal of Proteomics [1].

  5. A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

    Emilie Ernoult

    2010-01-01

    Full Text Available Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins.

  6. A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation.

    Ernoult, Emilie; Bourreau, Anthony; Gamelin, Erick; Guette, Catherine

    2010-01-01

    Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma "signatures," which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 microg of plasma proteins.

  7. iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

    Fátima Milhano Santos

    2018-04-01

    Full Text Available Rhegmatogenous retinal detachment (RRD is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1, glucose transporters (GLUT-1, growth factors (metalloproteinase inhibitor 1, and serine protease inhibitors (plasminogen activator inhibitor 1 are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

  8. Study of Nitrate Stress in Desulfovibrio vulgaris Hildenborough Using iTRAQ Proteomics

    Redding, A.M.; Mukhopadhyay, A.; Joyner, D.; Hazen, T.C.; Keasling, J.D.

    2006-10-12

    The response of Desulfovibrio vulgaris Hildenborough (DvH),a sulphate-reducing bacterium, to nitrate stress was examined usingquantitative proteomic analysis. DvH was stressed with 105 m M sodiumnitrate(NaNO3), a level that caused a 50 percent inhibition in growth.The protein profile of stressed cells was compared with that of cellsgrown in the absence of nitrate using the iTRAQ peptide labellingstrategy and tandem liquid chromatography separation coupled with massspectrometry (quadrupoletime-of-flight) detection. A total of 737 uniqueproteins were identified by two or more peptides, representing 22 percentof the total DvH proteome and spanning every functional category. Theresults indicate that this was a mild stress, as proteins involved incentral metabolism and the sulphate reduction pathway were unperturbed.Proteins involved in the nitrate reduction pathway increased. Increasesseen in transport systems for proline, glycine^ betaineandglutamateindicate that the NaNO3 exposure led to both salt stress and nitratestress.Up-regulation observed in oxidative stress response proteins (Rbr,RbO, etc.) and a large number of ABC transport systems as well as in iron^ sulphur -cluster-containing proteins, however, appear to be specific tonitrate exposure. Finally, a number of hypothetical proteins were amongthe most significant changers, indicating that there may be unknownmechanisms initiated upon nitrate stress in DvH.

  9. Quantitative proteomic analysis of serum from pregnant women carrying a fetus with conotruncal heart defect using isobaric tags for relative and absolute quantitation (iTRAQ labeling.

    Ying Zhang

    Full Text Available To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD fetus, using proteomic analysis.The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14-18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS. The differentially expressed proteins identified by iTRAQ were further validated with Western blot.A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.

  10. Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis.

    Bouchal, Pavel; Roumeliotis, Theodoros; Hrstka, Roman; Nenutil, Rudolf; Vojtesek, Borivoj; Garbis, Spiros D

    2009-01-01

    The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p biomarker discovery program.

  11. iTRAQ reveals proteomic changes during intestine regeneration in the sea cucumber Apostichopus japonicus.

    Sun, Lina; Xu, Dongxue; Xu, Qinzeng; Sun, Jingchun; Xing, Lili; Zhang, Libin; Yang, Hongsheng

    2017-06-01

    Sea cucumbers have a striking capacity to regenerate most of their viscera after evisceration, which has drawn the interest of many researchers. In this study, the isobaric tag for relative and absolute quantitation (iTRAQ) was utilized to investigate protein abundance changes during intestine regeneration in sea cucumbers. A total of 4073 proteins were identified, and 2321 proteins exhibited significantly differential expressions, with 1100 upregulated and 1221 downregulated proteins. Our results suggest that intestine regeneration constitutes a complex life activity regulated by the cooperation of various biological processes, including cytoskeletal changes, extracellular matrix (ECM) remodeling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Our results provided a basis for studying the regulatory mechanisms associated with sea cucumber regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots

    Abdallah Cosette

    2012-06-01

    Full Text Available Abstract Background Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests. Results In-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values. Conclusions The present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.

  13. iTRAQ proteomics analysis reveals that PI3K is highly associated with bupivacaine-induced neurotoxicity pathways.

    Zhao, Wei; Liu, Zhongjie; Yu, Xujiao; Lai, Luying; Li, Haobo; Liu, Zipeng; Li, Le; Jiang, Shan; Xia, Zhengyuan; Xu, Shi-yuan

    2016-02-01

    Bupivacaine, a commonly used local anesthetic, has potential neurotoxicity through diverse signaling pathways. However, the key mechanism of bupivacaine-induced neurotoxicity remains unclear. Cultured human SH-SY5Y neuroblastoma cells were treated (bupivacaine) or untreated (control) with bupivacaine for 24 h. Compared to the control group, bupivacaine significantly increased cyto-inhibition, cellular reactive oxygen species, DNA damage, mitochondrial injury, apoptosis (increased TUNEL-positive cells, cleaved caspase 3, and Bcl-2/Bax), and activated autophagy (enhanced LC3II/LC3I ratio). To explore changes in protein expression and intercommunication among the pathways involved in bupivacaine-induced neurotoxicity, an 8-plex iTRAQ proteomic technique and bioinformatics analysis were performed. Compared to the control group, 241 differentially expressed proteins were identified, of which, 145 were up-regulated and 96 were down-regulated. Bioinformatics analysis of the cross-talk between the significant proteins with altered expression in bupivacaine-induced neurotoxicity indicated that phosphatidyl-3-kinase (PI3K) was the most frequently targeted protein in each of the interactions. We further confirmed these results by determining the downstream targets of the identified signaling pathways (PI3K, Akt, FoxO1, Erk, and JNK). In conclusion, our study demonstrated that PI3K may play a central role in contacting and regulating the signaling pathways that contribute to bupivacaine-induced neurotoxicity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference.

    Zhu, Beibei; Li, Xiangyu; Chen, Huan; Wang, Hongjuan; Zhu, Xinchao; Hou, Hongwei; Hu, Qingyuan

    2017-05-13

    Repeated exposures to nicotine are known to result in persistent changes in proteins expression in addiction-related brain regions, such as the striatum, nucleus accumbens and prefrontal cortex, but the changes induced in the protein content of the hippocampus remain poorly studied. This study established a rat model of nicotine-induced conditioned place preference (CPP), and screened for proteins that were differentially expressed in the hippocampus of these rats using isobaric tags for relative and absolute quantitation labeling (iTRAQ) coupled with 2D-LC MS/MS. The nicotine-induced CPP was established by subcutaneously injecting rats with 0.2 mg/kg nicotine. Relative to the control (saline) group, the nicotine group showed 0.67- and 1.5-fold changes in 117 and 10 hippocampal proteins, respectively. These differentially expressed proteins are mainly involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function, long-term synaptic potentiation and nervous system development. Furthermore, RT-PCR was used to confirmed the results of the proteomic analysis. Our findings identify several proteins and cellular signaling pathways potentially involved in the molecular mechanisms in the hippocampus that underlie nicotine addiction. These results provide insights into the mechanisms of nicotine treatment in hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Strong, sudden cooling alleviates the inflammatory responses in heat-stressed dairy cows based on iTRAQ proteomic analysis

    Cheng, Jianbo; Min, Li; Zheng, Nan; Fan, Caiyun; Zhao, Shengguo; Zhang, Yangdong; Wang, Jiaqi

    2018-02-01

    This study was designed to investigate the effects of sudden cooling on the physiological responses of 12 heat-stressed Holstein dairy cows using an isobaric tags for relative and absolute quantification (iTRAQ) labeling approach. Plasma samples were collected from these cows during heat stress (HS), and after strong, sudden cooling in the summer (16 days later). We compared plasma proteomic data before and after sudden cooling to identify the differentially abundant proteins. The results showed that sudden cooling in summer effectively alleviated the negative consequences of HS on body temperature and production variables. Expressions of plasma hemoglobin alpha and hemoglobin beta were upregulated, whereas lipopolysaccharide-binding protein (LBP) and haptoglobin were downregulated in this process. The increase of hemoglobin after cooling may improve oxygen transport and alleviate the rise in respiration rates in heat-stressed dairy cows. The decrease of LBP and haptoglobin suggests that the inflammatory responses caused by HS are relieved after cooling. Our findings provide new insight into the physiological changes that occur when heat-stressed dairy cows experience strong, sudden cooling.

  16. Proteomic analysis of maize grain development using iTRAQ reveals temporal programs of diverse metabolic processes.

    Yu, Tao; Li, Geng; Dong, Shuting; Liu, Peng; Zhang, Jiwang; Zhao, Bin

    2016-11-04

    Grain development in maize is an essential process in the plant's life cycle and is vital for use of the plant as a crop for animals and humans. However, little is known regarding the protein regulatory networks that control grain development. Here, isobaric tag for relative and absolute quantification (iTRAQ) technology was used to analyze temporal changes in protein expression during maize grain development. Maize grain proteins and changes in protein expression at eight developmental stages from 3 to 50 d after pollination (DAP) were performed using iTRAQ-based proteomics. Overall, 4751 proteins were identified; 2639 of these were quantified and 1235 showed at least 1.5-fold changes in expression levels at different developmental stages and were identified as differentially expressed proteins (DEPs). The DEPs were involved in different cellular and metabolic processes with a preferential distribution to protein synthesis/destination and metabolism categories. A K-means clustering analysis revealed coordinated protein expression associated with different functional categories/subcategories at different development stages. Our results revealed developing maize grain display different proteomic characteristics at distinct stages, such as numerous DEPs for cell growth/division were highly expressed during early stages, whereas those for starch biosynthesis and defense/stress accumulated in middle and late stages, respectively. We also observed coordinated expression of multiple proteins of the antioxidant system, which are essential for the maintenance of reactive oxygen species (ROS) homeostasis during grain development. Particularly, some DEPs, such as zinc metallothionein class II, pyruvate orthophosphate dikinase (PPDK) and 14-3-3 proteins, undergo major changes in expression at specific developmental stages, suggesting their roles in maize grain development. These results provide a valuable resource for analyzing protein function on a global scale and also

  17. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

    Wang, Shuzhen; Chen, Wenyue; Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

    2015-01-01

    Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various

  18. Combined serum and EPS-urine proteomic analysis using iTRAQ technology for discovery of potential prostate cancer biomarkers.

    Zhang, Mo; Chen, Lizhu; Yuan, Zhengwei; Yang, Zeyu; Li, Yue; Shan, Liping; Yin, Bo; Fei, Xiang; Miao, Jianing; Song, Yongsheng

    2016-11-01

    Prostate cancer (PCa) is one of the most common malignant tumors and a major cause of cancer-related death for men worldwide. The aim of our study was to identify potential non-invasive serum and expressed prostatic secretion (EPS)-urine biomarkers for accurate diagnosis of PCa. Here, we performed a combined isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to compare protein profiles using pooled serum and EPS-urine samples from 4 groups of patients: benign prostate hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), localized PCa and metastatic PCa. The differentially expressed proteins were rigorously selected and further validated in a large and independent cohort using classical ELISA and Western blot assays. Finally, we established a multiplex biomarker panel consisting of 3 proteins (serum PF4V1, PSA, and urinary CRISP3) with an excellent diagnostic capacity to differentiate PCa from BPH [area under the receiver operating characteristic curve (AUC) of 0.941], which showed an evidently greater discriminatory ability than PSA alone (AUC, 0.757) (P<0.001). Importantly, even when PSA level was in the gray zone (4-10 ng/mL), a combination of PF4V1 and CRISP3 could achieve a relatively high diagnostic efficacy (AUC, 0.895). Furthermore, their combination also had the potential to distinguish PCa from HGPIN (AUC, 0.934). Our results demonstrated that the combined application of serum and EPS-urine biomarkers can improve the diagnosis of PCa and provide a new prospect for non-invasive PCa detection.

  19. A proteomics study of hyperhomocysteinemia injury of the hippocampal neurons using iTRAQ.

    Fang, Min; Wang, Jing; Yan, Han; Zhao, Yan-Xin; Liu, Xue-Yuan

    2014-11-01

    High levels of homocysteine, caused by abnormal methionine metabolism, can induce degeneration of mouse hippocampal neurons. iTRAQ™ technology has been widely used in the field of proteomics research and through employing this technology, the present study identified that hyperhomocysteinemia induced the downregulation of 52 proteins and upregulation of 44 proteins in the mouse hippocampus. Through gene ontology and pathway analysis, the upregulation of components of the cytoskeleton, actin, regulators of focal adhesion, calcium signaling pathways, tight junctions, ErbB and gonadotrophin‑releasing hormone signaling, leukocyte, transendothelial migration, propanoate and pyruvate metabolism, valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies and benzoate degradation via CoA ligation pathway, was identified. It was additionally verified that tau protein was highly expressed in the hyperhomocysteinemic neurons. Further analysis revealed that tau network proteins played functional roles in homocysteine‑induced neuronal damage.

  20. Development of quantitative proteomics using iTRAQ based on the immunological response of Galleria mellonella larvae challenged with Fusarium oxysporum microconidia.

    Amalia Muñoz-Gómez

    Full Text Available Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104

  1. Characterization and comparison of proteomes of albino sea cucumber Apostichopus japonicus (Selenka) by iTRAQ analysis.

    Xia, Chang-Ge; Zhang, Dijun; Ma, Chengnv; Zhou, Jun; He, Shan; Su, Xiu-Rong

    2016-04-01

    Sea cucumber is a commercially important marine organism in China. Of the different colored varieties sold in China, albino sea cucumber has the greatest appeal among consumers. Identification of factors contributing to albinism in sea cucumber is therefore likely to provide a scientific basis for improving the cultivability of these strains. In this study, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification labeling was used for the first time to quantitatively define the proteome of sea cucumbers and reveal proteomic characteristics unique to albino sea cucumbers. A total of 549 proteins were identified and quantified in albino sea cucumber and the functional annotations of 485 proteins have been exhibited based on COG database. Compared with green sea cucumber, 12 proteins were identified as differentially expressed in the intestine and 16 proteins in the body wall of albino sea cucumber. Among them, 5 proteins were up-regulated in the intestine and 8 proteins were down-regulated in body wall. Gene ontology annotations of these differentially expressed proteins consisted mostly of 'biological process'. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying albinism in sea cucumber. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Proteomics analysis of maize (Zea mays L.) grain based on iTRAQ reveals molecular mechanisms of poor grain filling in inferior grains.

    Yu, Tao; Li, Geng; Liu, Peng; Dong, Shuting; Zhang, Jiwang; Zhao, Bin

    2017-06-01

    In maize, inferior grains (IG) located on the upper part of the ear have poor grain filling process compared to superior grains (SG) located on the middle and lower parts of the ear. This difference limits satisfactory yield and quality; however, the underlying molecular mechanisms remain unknown. Here, using the isobaric tag for relative and absolute quantification (iTRAQ) technology, the proteomes of IG and SG during early and middle grain filling stages were investigated. In total, 4720 proteins were identified in maize grain and 305 differentially accumulated proteins (DiAPs) were detected between IG and SG. These DiAPs were involved in diverse cellular and metabolic processes with preferred distribution in protein synthesis/destination and metabolism. Compared to SG, DiAPs related to cell growth/division and starch synthesis were lag-accumulated and down-regulated in IG, respectively, resulting in smaller sink sizes and lower sink activities in IG. Meanwhile, impediment of the glycolysis pathway in IG may lead to reduce energy supply and building materials for substance synthesis. Additionally, reactive oxygen species (ROS) homeostasis and the defense system were disturbed in IG, which might lead to reduce protection against various environmental stresses. The present study provides new information on the proteomic differences between IG and SG, and explains possible molecular mechanisms for poor grain filling in IG. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

    Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen

    2012-11-02

    Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

  4. Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

    Liu, Pei

    2014-01-28

    Cellular redox status plays a key role in mediating various physiological and developmental processes often through modulating activities of redox-sensitive proteins. Various stresses trigger over-production of reactive oxygen/nitrogen species which lead to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. Here, we report a high-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols. The biotin-tagged peptides are affinity purified, labeled with iTRAQ reagents, and analyzed using a paralleled HCD-CID fragmentation mode in an LTQ-Orbitrap. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. This approach allows identification of the specific redox-regulated cysteine residues, and offers an effective tool for elucidation of redox proteomes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ

    Cui, Ruina; Zhang, Hongxia [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029 (China); Cui, Qianqian; Wang, Jianshe [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2015-12-15

    Highlights: • PFOS stimulates cell proliferation of human liver cell line (HL-7702). • Differential expressed proteins are identified by iTRAQ. • Most of differential proteins caused by PFOS are related to cell proliferation. • Up-regulation of cyclin/cdk by PFOS plays a role in driving cells into cell cycle. - Abstract: Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50 μM PFOS for 48 h and 96 h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200 μM) stimulated HL-7702 cell viability at 48 h and 96 h, reduced the G0/G1 percentage, and increased the S + G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

  6. Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ

    Cui, Ruina; Zhang, Hongxia; Guo, Xuejiang; Cui, Qianqian; Wang, Jianshe; Dai, Jiayin

    2015-01-01

    Highlights: • PFOS stimulates cell proliferation of human liver cell line (HL-7702). • Differential expressed proteins are identified by iTRAQ. • Most of differential proteins caused by PFOS are related to cell proliferation. • Up-regulation of cyclin/cdk by PFOS plays a role in driving cells into cell cycle. - Abstract: Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50 μM PFOS for 48 h and 96 h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200 μM) stimulated HL-7702 cell viability at 48 h and 96 h, reduced the G0/G1 percentage, and increased the S + G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

  7. Differential Proteomic Analysis of Syncytiotrophoblast Extracellular Vesicles from Early-Onset Severe Preeclampsia, using 8-Plex iTRAQ Labeling Coupled with 2D Nano LC-MS/MS

    Hongmei Li

    2015-06-01

    Full Text Available Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM may lead to preeclampsia (PE. We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8 related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE.

  8. Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

    2007-12-01

    that fascinating fungus known as Coccidioides. I also want to thank the UA Mass Spectrometry Facility and the UA Proteomics Consortium, especially...W. & N. N. Kav. 2006. The proteome of the phytopathogenic fungus Sclerotinia sclerotiorum. Proteomics 6: 5995-6007. 127. de Godoy, L. M., J. V...IDENTIFICATION OF PROTEIN VACCINE CANDIDATES USING COMPREHENSIVE PROTEOMIC ANALYSIS STRATEGIES by James G. Rohrbough

  9. Relative quantitation of proteins fractionated by the ProteomeLabTM PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ)

    Skalníková, Helena; Řehulka, Pavel; Chmelík, Josef; Martinková, Jiřina; Zilvarová, Michaela; Gadher, S. J.; Kovářová, Hana

    2007-01-01

    Roč. 389, č. 5 (2007), s. 1639-1645 ISSN 1618-2642 R&D Projects: GA ČR GA301/05/0418; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z40310501 Keywords : 2D liquid chromatography * human T-lymphoblastic leukemia cell proteins * iTRAQ Subject RIV: CE - Biochemistry Impact factor: 2.867, year: 2007

  10. Identification of New Epididymal Luminal Fluid Proteins Involved in Sperm Maturation in Infertile Rats Treated by Dutasteride Using iTRAQ

    Shu-Wu Xie

    2016-05-01

    Full Text Available Background: Spermatozoa become mature and acquire fertilizing capacity during their passage through the epididymal lumen. In this study, we identified new epididymal luminal fluid proteins involved in sperm maturation in infertile rats by dutasteride, a dual 5α-reductase inhibitor, in order to provide potential epididymal targets for new contraceptives and infertility treatment. Methods: Male rats were treated with dutasteride for 28 consecutive days. We observed the protein expression profiles in the epididymal luminal fluids in infertile and normal rats using isobaric tags for relative and absolute quantitation (iTRAQ technique. The confidence of proteome data was validated by enzyme-linked immunosorbent assays. Results: 1045 proteins were tested, and 23 of them presented different expression profiling in the infertile and normal rats. The seven proteins were down-regulated, and 16 proteins were up-regulated. Among the seven proteins which were significantly down-regulated by dutasteride in the epididymal luminal fluids, there were three β-defensins (Defb2, Defb18 and Defb39, which maybe the key proteins involved in epididymal sperm maturation and male fertility. Conclusions: We report for the first time that dutasteride influences the protein expression profiling in the epididymal luminal fluids of rats, and this result provides some new epididymal targets for male contraception and infertility therapy.

  11. Proteomic comparison by iTRAQ combined with mass spectrometry of egg white proteins in laying hens (Gallus gallus) fed with soybean meal and cottonseed meal

    He, Tao; Zhang, Haijun; Wang, Jing; Wu, Shugeng; Yue, Hongyuan; Qi, Guanghai

    2017-01-01

    Cottonseed meal (CSM) is commonly used in hens’ diets to replace soybean meal (SBM). However, the molecular consequences of this substitution remains unclear. To investigate the impact of this substitution at the molecular level, iTRAQ combined with biochemical analysis was performed in Hy-Line W-36 hens supplemented with a mixed diet of CSM and SBM. Egg weight, albumen height, and Haugh unit were significantly reduced in the CSM100 group (100% crude protein of SBM replaced by CSM) compared with the SBM group (Phen diet. PMID:28813468

  12. Comparative proteomic analysis of soybean leaves and roots by iTRAQ provides insights into response mechanisms to short-term salt stress

    Wei eJi

    2016-04-01

    Full Text Available Salinity severely threatens land use capability and crop yields worldwide. Understanding the mechanisms that protect soybean from salt stress will help in the development of salt-stress tolerant leguminous plants. Here we firstly analyzed the changes in malondialdehyde levels, the activities of superoxide dismutase and peroxidases, cholorophyll contents, and Na+/K+ ratios in leaves and roots from soybean seedlings treated with 200 mM NaCl for different time points, and suggested that 200 mM NaCl treated for 12 h was enough for exploring proteomic analysis to soybean seedlings. iTRAQ-based proteomic approach was used to investigate the proteomes of soybean leaves and roots under salt treatment. Data are available via ProteomeXchange with identifier PXD002851. In total, 278 and 440 proteins with significantly altered abundance were identified in leaves and roots of soybean, respectively, with only 50 mutual unique proteins in the both tissues. These identified differentially expressed proteins (DEPs were mainly involved in 13 biological processes. Moreover, protein-protein interaction analysis revealed that the proteins involved in metabolism, carbohydrate and energy metabolism, protein synthesis and redox homeostasis constructed four types of response networks to high salt stress. Besides, semi-quantitative RT-PCR analysis revealed that some of the proteins, such as 14-3-3, MMK2, PP1, TRX-h, were also regulated by salt stress at the level of transcription. These results indicated that effective regulatory protein expression related to signalling, membrane and transport, stress defense and metabolism played important roles in the short-term salt response of soybean seedlings.

  13. Proteomics: Protein Identification Using Online Databases

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  14. Global Liver Proteome Analysis Using iTRAQ Reveals AMPK-mTOR-Autophagy Signaling Is Altered by Intrauterine Growth Restriction in Newborn Piglets.

    Long, Baisheng; Yin, Cong; Fan, Qiwen; Yan, Guokai; Wang, Zhichang; Li, Xiuzhi; Chen, Changqing; Yang, Xingya; Liu, Lu; Zheng, Zilong; Shi, Min; Yan, Xianghua

    2016-04-01

    Intrauterine growth restriction (IUGR) impairs fetal growth and development, perturbs nutrient metabolism, and increases the risk of developing diseases in postnatal life. However, the underlying mechanisms by which IUGR affects fetal liver development and metabolism remain incompletely understood. Here, we applied a high-throughput proteomics approach and biochemical analysis to investigate the impact of IUGR on the liver of newborn piglets. As a result, we identified 78 differentially expressed proteins in the three biological replicates, including 31 significantly up-regulated proteins and 47 significantly down-regulated proteins. Among them, a majority of differentially expressed proteins were related to nutrient metabolism and mitochondrial function. Additionally, many significantly down-regulated proteins participated in the mTOR signaling pathway and the phagosome maturation signaling pathway. Further analysis suggested that glucose concentration and hepatic glycogen storage were both reduced in IUGR newborn piglets, which may contribute to AMPK activation and mTORC1 inhibition. However, AMPK activation and mTORC1 inhibition failed to induce autophagy in the liver of IUGR neonatal pigs. A possible reason is that PP2Ac, a potential candidate in autophagy regulation, is significantly down-regulated in the liver of IUGR newborn piglets. These findings may provide implications for preventing and treating IUGR in human beings and domestic animals.

  15. Proteome identification of the silkworm middle silk gland

    Jian-ying Li

    2016-03-01

    Full Text Available To investigate the functional differentiation among the anterior (A, middle (M, and posterior (P regions of silkworm middle silk gland (MSG, their proteomes were characterized by shotgun LC–MS/MS analysis with a LTQ-Orbitrap mass spectrometer. To get better proteome identification and quantification, triplicate replicates of mass spectrometry analysis were performed for each sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014 [1] via the PRIDE partner repository (Vizcaino, 2013 [2] with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003371. The peptide identifications that were further processed by PeptideProphet program in Trans-Proteomic Pipeline (TPP after database search with Mascot software were also available in .XML format files. Data presented here are related to a research article published in Journal of Proteomics by Li et al. (2015 [3]. Keywords: Bombyx mori, Middle silk gland, Silk protein synthesis, Shotgun proteomics, Label-free

  16. Describing the Diapause-Preparatory Proteome of the Beetle Colaphellus bowringi and Identifying Candidates Affecting Lipid Accumulation Using Isobaric Tags for Mass Spectrometry-Based Proteome Quantification (iTRAQ

    Daniel A. Hahn

    2017-04-01

    Full Text Available Prior to entering diapause, insects must prepare themselves physiologically to withstand the stresses of arresting their development for a lengthy period. While studies describing the biochemical and cellular milieu of the maintenance phase of diapause are accumulating, few studies have taken an “omics” approach to describing molecular events during the diapause preparatory phase. We used isobaric tags and mass spectrometry (iTRAQ to quantitatively compare the expression profiles of proteins identified during the onset of diapause preparation phase in the heads of adult female cabbage beetles, Colaphellus bowringi. A total of 3,175 proteins were identified, 297 of which were differentially expressed between diapause-destined and non-diapause-destined female adults and could therefore be involved in diapause preparation in this species. Comparison of identified proteins with protein function databases shows that many of these differentially expressed proteins enhanced in diapause destined beetles are involved in energy production and conversion, carbohydrate metabolism and transport, and lipid metabolism. Further hand annotation of differentially abundant peptides nominates several associated with stress hardiness, including HSPs and antioxidants, as well as neural development. In contrast, non-diapause destined beetles show substantial increases in cuticle proteins, suggesting additional post-emergence growth. Using RNA interference to silence a fatty acid-binding protein (FABP that was highly abundant in the head of diapause-destined females prevented the accumulation of lipids in the fat body, a common product of diapause preparation in this species and others. Surprisingly, RNAi against the FABP also affected the transcript abundance of several heat shock proteins. These results suggest that the identified differentially expressed proteins that play vital roles in lipid metabolism may also contribute somehow to enhanced hardiness to

  17. Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation

    Lücker, Joost; Laszczak, Mario; Smith, Derek; Lund, Steven T

    2009-01-01

    Background iTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB. Results We were interested in characterizing ripening initiation ('veraison') in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level. Conclusion We determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening initiation and may be further

  18. Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon berries at ripening initiation

    Smith Derek

    2009-01-01

    Full Text Available Abstract Background iTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB. Results We were interested in characterizing ripening initiation ('veraison' in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level. Conclusion We determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening

  19. Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling

    Su, Dian; Gaffrey, Matthew J.; Guo, Jia; Hatchell, Kayla E.; Chu, Rosalie K.; Clauss, Therese RW; Aldrich, Joshua T.; Wu, Si; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.; Thrall, Brian D.; Qian, Weijun

    2014-02-11

    Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with free radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.

  20. Proteomic identification of gender molecular markers in Bothrops jararaca venom.

    Zelanis, André; Menezes, Milene C; Kitano, Eduardo S; Liberato, Tarcísio; Tashima, Alexandre K; Pinto, Antonio F M; Sherman, Nicholas E; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

    2016-04-29

    Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being

  1. A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

    Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

    2017-08-15

    Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Identification and characterization of N-glycosylated proteins using proteomics

    Selby, David S; Larsen, Martin R; Calvano, Cosima Damiana

    2008-01-01

    and analysis of glycoproteins and glycopeptides. Combinations of affinity-enrichment techniques, chemical and biochemical protocols, and advanced mass spectrometry facilitate detailed glycoprotein analysis in proteomics, from fundamental biological studies to biomarker discovery in biomedicine....... is a complex task and is currently achieved by mass spectrometry-based methods that enable identification of glycoproteins and localization, classification, and analysis of individual glycan structures on proteins. In this chapter we briefly introduce a range of analytical technologies for recovery...

  3. iTRAQ analysis of hepatic proteins in free-living Mus spretus mice to assess the contamination status of areas surrounding Doñana National Park (SW Spain)

    Abril, Nieves; Chicano-Gálvez, Eduardo; Michán, Carmen; Pueyo, Carmen; López-Barea, Juan, E-mail: bb1lobaj@uco.es

    2015-08-01

    This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥ 2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas. - Highlights: • iTRAQ quantitation was used for the first time to monitor a wildlife reserve • Over 2,000 proteins with altered expression were identified in problem Doñana sites • Of them, 118 changed over 2.5-fold in, at least, two problem sites • Upregulation of protective proteins

  4. Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

    Welker, F

    2018-02-20

    The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identifications at increased evolutionary distances due to a larger number of protein sequence differences between the database sequence and the analyzed organism. Error-tolerant proteomic search algorithms should theoretically overcome this problem at both the peptide and protein level; however, this has not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against sequence databases at increasing evolutionary distances: the human (0 Ma), chimpanzee (6-8 Ma) and orangutan (16-17 Ma) reference proteomes, respectively. Incorrectly suggested amino acid substitutions are absent when employing adequate filtering criteria for mutable Peptide Spectrum Matches (PSMs), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations between the target and database sequences are the main factors influencing mutable PSM identification. The error-tolerant results suggest that the cross-species proteomics problem is not overcome at increasing evolutionary distances, even at the protein level. Peptide and protein loss has the potential to significantly impact divergence dating and proteome comparisons when using ancient samples as there is a bias towards the identification of conserved sequences and proteins. Effects are minimized

  5. Identification of Proteins Using iTRAQ and Virus-Induced Gene Silencing Reveals Three Bread Wheat Proteins Involved in the Response to Combined Osmotic-Cold Stress.

    Zhang, Ning; Zhang, Lingran; Shi, Chaonan; Zhao, Lei; Cui, Dangqun; Chen, Feng

    2018-05-25

    Crops are often subjected to a combination of stresses in the field. To date, studies on the physiological and molecular responses of common wheat to a combination of osmotic and cold stresses, however, remain unknown. In this study, wheat seedlings exposed to osmotic-cold stress for 24 h showed inhibited growth, as well as increased lipid peroxidation, relative electrolyte leakage, and soluble sugar contents. iTRAQ-based quantitative proteome method was employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to osmotic-cold stress conditions. A total of 250 and 258 proteins with significantly altered abundance in the roots and leaves were identified, respectively, and the majority of these proteins displayed differential abundance, thereby revealing organ-specific differences in adaptation to osmotic-cold stress. Yeast two hybrid assay examined five pairs of stress/defense-related protein-protein interactions in the predicted protein interaction network. Furthermore, quantitative real-time PCR analysis indicated that abiotic stresses increased the expression of three candidate protein genes, i.e., TaGRP2, CDCP, and Wcor410c in wheat leaves. Virus-induced gene silencing indicated that three genes TaGRP2, CDCP, and Wcor410c were involved in modulating osmotic-cold stress in common wheat. Our study provides useful information for the elucidation of molecular and genetics bases of osmotic-cold combined stress in bread wheat.

  6. Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment

    Welker, F.

    2018-01-01

    Background: The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identificati......Background: The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein...... not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against......), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations...

  7. Identification of SUMO conjugation sites in the budding yeast proteome

    Miguel Esteras

    2017-10-01

    Full Text Available Post-translational modification by the small ubiquitin-like modifier (SUMO is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

  8. Enhanced detection method for corneal protein identification using shotgun proteomics

    Schlager John J

    2009-06-01

    Full Text Available Abstract Background The cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea. Results Rabbit corneas were purchased, the epithelium and endothelium regions were removed, proteins processed and separately analyzed using liquid chromatography/mass spectrometry. Proteins identified from separate layers were compared against results from complete corneal samples. Protein digests were separated using a six hour liquid chromatographic gradient and ion-trap mass spectrometry used for detection of eluted peptide fractions. The SEQUEST database search results were filtered to allow only proteins with match probabilities of equal or better than 10-3 and peptides with a probability of 10-2 or less with at least two unique peptides isolated within

  9. Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

    Rohrbough, James G

    2007-01-01

    Presented in this dissertation are proteomic analysis studies focused on identifying proteins to be used as vaccine candidates against Coccidioidomycosis, a potentially fatal human pulmonary disease...

  10. Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.).

    Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Pathak, Dharminder; Chen, Sixue; Wendel, Jonathan F

    2014-12-01

    Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC-MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30% of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.

  11. Comparative Proteomic Profiling and Biomarker Identification of Traditional Chinese Medicine-Based HIV/AIDS Syndromes.

    Wen, Li; Liu, Ye-Fang; Jiang, Cen; Zeng, Shao-Qian; Su, Yue; Wu, Wen-Jun; Liu, Xi-Yang; Wang, Jian; Liu, Ying; Su, Chen; Li, Bai-Xue; Feng, Quan-Sheng

    2018-03-08

    Given the challenges in exploring lifelong therapy with little side effect for human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) cases, there is increasing interest in developing traditional Chinese medicine (TCM) treatments based on specific TCM syndrome. However, there are few objective and biological evidences for classification and diagnosis of HIV/AIDS TCM syndromes to date. In this study, iTRAQ-2DLC-MS/MS coupled with bioinformatics were firstly employed for comparative proteomic profiling of top popular TCM syndromes of HIV/AIDS: accumulation of heat-toxicity (AHT) and Yang deficiency of spleen and kidney (YDSK). It was found that for the two TCM syndromes, the identified differential expressed proteins (DEPs) as well as their biological function distributions and participation in signaling pathways were significantly different, providing biological evidence for the classification of HIV/AIDS TCM syndromes. Furthermore, the TCM syndrome-specific DEPs were confirmed as biomarkers based on western blot analyses, including FN1, GPX3, KRT10 for AHT and RBP4, ApoE, KNG1 for YDSK. These biomarkers also biologically linked with the specific TCM syndrome closely. Thus the clinical and biological basis for differentiation and diagnosis of HIV/AIDs TCM syndromes were provided for the first time, providing more opportunities for stable exertion and better application of TCM efficacy and superiority in HIV/AIDS treatment.

  12. Proteomic platform for the identification of proteins in olive (Olea europaea) pulp.

    Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Piovesana, Susy; Samperi, Roberto; Stampachiacchiere, Serena; Laganà, Aldo

    2013-10-24

    The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues. Copyright © 2013. Published by Elsevier B.V.

  13. Target identification of natural and traditional medicines with quantitative chemical proteomics approaches.

    Wang, Jigang; Gao, Liqian; Lee, Yew Mun; Kalesh, Karunakaran A; Ong, Yong Siang; Lim, Jaehong; Jee, Joo-Eun; Sun, Hongyan; Lee, Su Seong; Hua, Zi-Chun; Lin, Qingsong

    2016-06-01

    Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines. Copyright © 2016. Published by Elsevier Inc.

  14. Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources

    Sunil S. Adav

    2015-06-01

    Full Text Available Here, we provide data related to the research article entitled “Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes” by Adav et al. (J. Proteomics (2015 [1]. Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ. The data presented here represents the detailed comparative abundances of diverse groups of biomass hydrolyzing enzymes including cellulases, hemicellulases, lignin degrading enzymes, and peptidases and proteases; and their post translational modification like deamidation.

  15. Global Proteome Analysis of Leptospira interrogans

    Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometr...

  16. Proteomic identification of CIB1 as a potential diagnostic factor in ...

    Proteomic identification of CIB1 as a potential diagnostic factor in hepatocellular carcinoma. Tong Junrong Zhou Huancheng H E Feng Gao Yi Yang Xiaoqin Luo Zhengmao Zhang Hong Zeng Jianying Wang Yin Huang Yuanhang Zhang Jianlin Sun Longhua He Guolin. Articles Volume 36 Issue 4 September 2011 pp 659- ...

  17. Proteomic identification of rhythmic proteins in rice seedlings.

    Hwang, Heeyoun; Cho, Man-Ho; Hahn, Bum-Soo; Lim, Hyemin; Kwon, Yong-Kook; Hahn, Tae-Ryong; Bhoo, Seong Hee

    2011-04-01

    Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes. 2011 Elsevier B.V. All rights reserved.

  18. Identification of Biomarkers for Endometriosis Using Clinical Proteomics

    Yang Zhao

    2015-01-01

    Full Text Available Background: We investigated possible biomarkers for endometriosis (EM using the ClinProt technique and proteomics methods. Methods: We enrolled 50 patients with EM, 34 with benign ovarian neoplasms and 40 healthy volunteers in this study. Serum proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS combined with weak cationic exchange (WCX magnetic beads. Possible biomarkers were analyzed by a random and repeat pattern model-validation method that we designed, and ClinProtools software, results were refined using online liquid chromatography-tandem MS. Results: We found a cluster of 5 peptides (4210, 5264, 2660, 5635, and 5904 Da, using 3 peptides (4210, 5904, 2660 Da to discriminate EM patients from healthy volunteers, with 96.67% sensitivity and 100% specificity. We selected 4210 and 5904 m/z, which differed most between patients with EM and controls, and identified them as fragments of ATP1B4, and the fibrinogen alpha (FGA isoform 1/2 of the FGA chain precursor, respectively. Conclusions: ClinProt can identify EM biomarkers, which - most notably - distinguish even early-stage or minimal disease. We found 5 stable peaks at 4210, 5264, 2660, 5635, and 5904 Da as potential EM biomarkers, the strongest of which were associated with ATP1B4 (4210 Da and FGA (5904 Da; this indicates that ATP1B4 and FGA are associated with EM pathogenesis.

  19. Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria.

    Cheng, Keding; Chui, Huixia; Domish, Larissa; Hernandez, Drexler; Wang, Gehua

    2016-04-01

    Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS-based diagnosis methods for bacteria identification and typing have been created, not only on well-accepted MALDI-TOF-MS-based fingerprint matches, but also on solving the insufficiencies of MALDI-TOF-MS-based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS-based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria. © 2016 The Authors. PROTEOMICS - Clinical Applications Published by WILEY-VCH Verlag GmbH & Co. KGaA.

  20. Identification and proteomic analysis of osteoblast-derived exosomes

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

  1. Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography—Tandem Mass Spectrometry

    Tabb, David L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Variyath, Asokan Mulayath; Ham, Amy-Joan L.; Bunk, David M.; Kilpatrick, Lisa E.; Billheimer, Dean D.; Blackman, Ronald K.; Cardasis, Helene L.; Carr, Steven A.; Clauser, Karl R.; Jaffe, Jacob D.; Kowalski, Kevin A.; Neubert, Thomas A.; Regnier, Fred E.; Schilling, Birgit; Tegeler, Tony J.; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fisher, Susan J.; Gibson, Bradford W.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Steven E.; Tempst, Paul; Paulovich, Amanda G.; Liebler, Daniel C.; Spiegelman, Cliff

    2009-01-01

    The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind

  2. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

    Barkla, Bronwyn J.

    2016-01-01

    Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may cont...

  3. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Proteomic identification of secreted proteins of Propionibacterium acnes

    Holland Carsten

    2010-08-01

    Full Text Available Abstract Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS. A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence

  5. Unrestrictive identification of post-translational modifications in the urine proteome without enrichment

    2013-01-01

    Background Research on the human urine proteome may lay the foundation for the discovery of relevant disease biomarkers. Post-translational modifications (PTMs) have important effects on the functions of protein biomarkers. Identifying PTMs without enrichment adds no extra steps to conventional identification procedures for urine proteomics. The only difference is that this method requires software that can conduct unrestrictive identifications of PTMs. In this study, routine urine proteomics techniques were used to identify urine proteins. Unspecified PTMs were searched by MODa and PEAKS 6 automated software, followed by a manual search to screen out in vivo PTMs by removing all in vitro PTMs and amino acid substitutions. Results There were 75 peptides with 6 in vivo PTMs that were found by both MODa and PEAKS 6. Of these, 34 peptides in 18 proteins have novel in vivo PTMs compared with the annotation information of these proteins on the Universal Protein Resource website. These new in vivo PTMs had undergone methylation, dehydration, oxidation, hydroxylation, phosphorylation, or dihydroxylation. Conclusions In this study, we identified PTMs of urine proteins without the need for enrichment. Our investigation may provide a useful reference for biomarker discovery in the future. PMID:23317149

  6. Identification of cancer protein biomarkers using proteomic techniques

    Mor, Gil G.; Ward, David C.; Bray-Ward, Patricia

    2016-10-18

    The claimed invention describes methods to diagnose or aid in the diagnosis of cancer. The claimed methods are based on the identification of biomarkers which are particularly well suited to discriminate between cancer subjects and healthy subjects. These biomarkers were identified using a unique and novel screening method described herein. The biomarkers identified herein can also be used in the prognosis and monitoring of cancer. The invention comprises the use of leptin, prolactin, OPN and IGF-II for diagnosing, prognosis and monitoring of ovarian cancer.

  7. Genomic and proteomic identification of Late Holocene remains

    Biard, Vincent; Gol'din, Pavel; Gladilina, Elena

    2017-01-01

    A critical challenge of the 21st century is to understand and minimise the effects of human activities on biodiversity. Cetaceans are a prime concern in biodiversity research, as many species still suffer from human impacts despite decades of management and conservation efforts. Zooarchaeology...... sequencing approach. In addition, shotgun sequencing produced several complete ancient odontocete mitogenomes and auxiliary nuclear genomic data for further exploration in a population genetic context. In contrast, both morphological identification and Sanger sequencing lacked taxonomic resolution and....../or resulted in misclassification of samples. We found that the combination of ZooMS and shotgun sequencing provides a powerful tool in zooarchaeology, and here allowed for a deeper understanding of past marine resource use and its implication for current management and conservation of Black Sea odontocetes....

  8. Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

    Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

    2018-05-01

    The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Proteomic analysis of grapevine resistance induced by Trichoderma harzianum T39 reveals specific defence pathways activated against downy mildew

    Perazzolli, Michele

    2012-01-01

    Downy mildew is caused by the oomycete Plasmopara viticola and is one of the most serious diseases of grapevine. The beneficial microorganism Trichoderma harzianum T39 (T39) has previously been shown to induce plant-mediated resistance and to reduce the severity of downy mildew in susceptible grapevines. In order to better understand the cellular processes associated with T39-induced resistance, the proteomic and histochemical changes activated by T39 in grapevine were investigated before and 1 day after P. viticola inoculation. A comprehensive proteomic analysis of T39-induced resistance in grapevine was performed using an eight-plex iTRAQ protocol, resulting in the identification and quantification of a total of 800 proteins. Most of the proteins directly affected by T39 were found to be involved in signal transduction, indicating activation of a complete microbial recognition machinery. Moreover, T39-induced resistance was associated with rapid accumulation of reactive oxygen species and callose at infection sites, as well as changes in abundance of proteins involved in response to stress and redox balance, indicating an active defence response to downy mildew. On the other hand, proteins affected by P. viticola in control plants mainly decreased in abundance, possibly reflecting the establishment of a compatible interaction. Finally, the high-throughput iTRAQ protocol allowed de novo peptide sequencing, which will be used to improve annotation of the Vitis vinifera cv. Pinot Noir proteome. PMID:23105132

  10. Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

    Honoré Bent

    2010-01-01

    Full Text Available Abstract The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and mass spectrometry (MS. Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.

  11. A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

    Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Mu, Zheng; Jennings, Jennifer L; Hoek, Kristen L; Allos, Tara; Howard, Leigh M; Edwards, Kathryn M; Weil, P Anthony; Link, Andrew J

    2013-03-01

    Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

  12. Proteomics

    Tølbøll, Trine Højgaard; Danscher, Anne Mette; Andersen, Pia Haubro

    2012-01-01

    to current research strategies there is a need to develop novel approaches and methods that expand understanding of the disease mechanisms involved in CHD. The objectives of the present study were to explore the potential of liquid chromatography tandem mass spectrometry (LC–MS/MS) in mapping protein...... expression in three different bovine claw tissues, and to provide a relevant functional annotation of the proteins characterized in these tissues. LC–MS/MS was used to characterize protein expression in coronary band skin (C), claw dermal (D) and lamellar (L) tissues from two heifers. A total of 388...... different proteins were identified, with 146 proteins available for identification in C, 279 proteins in D and 269 proteins in L. A functional annotation of the identified proteins was obtained using the on-line Blast2GO tool. Three hundred and sixteen of the identified proteins could be subsequently...

  13. Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

    Verma, Shailender Kumar; Sharma, Ankita; Sandhu, Padmani; Choudhary, Neha; Sharma, Shailaja; Acharya, Vishal; Akhter, Yusuf

    2017-05-01

    Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Proteomics - a novel approach to the identification and characterisation of plasmodesmatal proteins

    Faulkner, C.R.; Blackman, L.M.; Lyon, B.R.; Overall, R.L.

    2001-01-01

    The development of proteomic methods, such as 2-dimensional gel electrophoresis (2-DE), has established a high resolution means of identifying and characterising proteins from a given protein mixture. The biochemical composition of plasmodesmata, the intercellular channels between plant cells, is poorly described despite extensive attempts to identify protemaceous plasmodesmatal components. These attempts have been confounded by the large number of proteins in the cell wall. We have exploited the anatomy of the alga Chara corallina to separate tissues with (nodal cells) and tissues without (internodal cells) plasmodesmata. Proteins specific to the cytoplasmic and wall protein extracts of nodal and internodal tissue were identified by comparison of 2-DE gels of these extracts. In particular, a 95 kDa protein was identified as specific to the nodal cells in both 1-dimensional and 2-dimensional comparisons of cytoplasmic nodal and internodal protein extracts. This protein was analysed by electron spray ionization time of flight tandem mass spectroscopy (ESI-TOF MS/MS) and the sequence obtained showed similarity to plant lipoxygenases. Further proteins of interest were identified in 2-DE resolution of extracts from the nodal cytoplasm, including two 49 kDa proteins and two 46 kDa proteins, and from the nodal cell walls, including a cluster of proteins around 30 kDa. Thus, a proteomic strategy for the identification and characterisation of proteins specific to different cell types in Chara corallina has been developed, with potential application to the identification and characterisation of plasmodesmatal proteins

  15. Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

    Liu, Pei; Zhang, Huoming; Wang, Hai; Xia, Yiji

    2014-01-01

    -throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential

  16. Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters.

    Sloothaak, J; Odoni, D I; de Graaff, L H; Martins Dos Santos, V A P; Schaap, P J; Tamayo-Ramos, J A

    2015-01-01

    The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger

  17. Identification of Novel Translational Urinary Biomarkers for Acetaminophen-Induced Acute Liver Injury Using Proteomic Profiling in Mice

    van Swelm, Rachel P. L.; Laarakkers, Coby M. M.; van der Kuur, Ellen C.; Morava-Kozicz, Eva; Wevers, Ron A.; Augustijn, Kevin D.; Touw, Daan J.; Sandel, Maro H.; Masereeuw, Rosalinde; Russel, Frans G. M.

    2012-01-01

    Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced

  18. Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.

    Mehdi Mesri

    Full Text Available Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

  19. Proteomic identification of altered cerebral proteins in the complex regional pain syndrome animal model.

    Nahm, Francis Sahngun; Park, Zee-Yong; Nahm, Sang-Soep; Kim, Yong Chul; Lee, Pyung Bok

    2014-01-01

    Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

  20. Proteomic Identification of Altered Cerebral Proteins in the Complex Regional Pain Syndrome Animal Model

    Francis Sahngun Nahm

    2014-01-01

    Full Text Available Background. Complex regional pain syndrome (CRPS is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

  1. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity.

    Barkla, Bronwyn J

    2016-09-08

    Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised.

  2. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

    Bronwyn J. Barkla

    2016-09-01

    Full Text Available Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised.

  3. Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria

    Huang Honglei

    2012-05-01

    Full Text Available Abstract Background Malaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear. Methods A protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins. A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive–malaria slide and compared with that of 17 malaria-negative children with fever. Results The proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification. Conclusions This study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

  4. Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

    Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

    2015-01-15

    Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

  5. Looking for prosocial genes: ITRAQ analysis of proteins involved in MDMA-induced sociability in mice.

    Kuteykin-Teplyakov, Konstantin; Maldonado, Rafael

    2014-11-01

    Social behavior plays a fundamental role in life of many animal species, allowing the interaction between individuals and sharing of experiences, needs, and goals across them. In humans, some neuropsychiatric diseases, including anxiety, posttraumatic stress disorder and autism spectrum disorders, are often characterized by impaired sociability. Here we report that N-Methyl-3,4-methylenedioxyamphetamine (MDMA, "Ecstasy") at low dose (3mg/kg) has differential effects on mouse social behavior. In some animals, MDMA promotes sociability without hyperlocomotion, whereas in other mice it elevates locomotor activity without affecting sociability. Both WAY-100635, a selective antagonist of 5-HT1A receptor, and L-368899, a selective oxytocin receptor antagonist, abolish prosocial effects of MDMA. Differential quantitative analysis of brain proteome by isobaric tag for relative and absolute quantification technology (iTRAQ) revealed 21 specific proteins that were highly correlated with sociability, and allowed to distinguish between entactogenic prosocial and hyperlocomotor effects of MDMA on proteome level. Our data suggest particular relevance of neurotransmission mediated by GABA B receptor, as well as proteins involved in energy maintenance for MDMA-induced sociability. Functional association network for differentially expressed proteins in cerebral cortex, hippocampus and amygdala were identified. These results provide new information for understanding the neurobiological substrate of sociability and may help to discover new therapeutic approaches to modulate social behavior in patients suffering from social fear and low sociability. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  6. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

    Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

    2010-08-06

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

  7. Quantitative analysis of UV-A shock and short term stress using iTRAQ, pseudo selective reaction monitoring (pSRM) and GC-MS based metabolite analysis of the cyanobacterium Nostoc punctiforme ATCC 29133.

    Wase, Nishikant; Pham, Trong Khoa; Ow, Saw Yen; Wright, Phillip C

    2014-09-23

    A quantitative proteomics and metabolomics analysis was performed using iTRAQ, HPLC and GC-MS in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 to understand the effect of short and long term UV-A exposure. Changes in the proteome were measured for short-term stress (4-24h) using iTRAQ. Changes in the photosynthetic pigments and intracellular metabolites were observed at exposures of up to 7days (pigments) and up to 11days (intracellular metabolites). To assess iTRAQ measurement quality, pseudo selected reaction monitoring (pSRM) was used, with this confirming underestimation of protein abundance levels by iTRAQ. Our results suggest that short term UV-A radiation lowers the abundance of PS-I and PS-II proteins. We also observed an increase in abundance of intracellular redox homeostasis proteins and plastocyanin. Additionally, we observed statistically significant changes in scytonemin, Chlorophyll A, astaxanthin, zeaxanthin, and β-carotene. Assessment of intracellular metabolites showed significant changes in several, suggesting their potential role in the Nostoc's stress mitigation strategy. Cyanobacteria under UV-A radiation have reduced growth due to intensive damage to essential functions, but the organism shows a defense response by remodeling bioenergetics pathway, induction of the UV protection compound scytonemin and increased levels of proline and tyrosine as a mitigation response. The effect of UV-A radiation on the proteome and intracellular metabolites of N. punctiforme ATCC 29133 including photosynthetic pigments has been described. We also verify the expression of 13 iTRAQ quantified protein using LC-pSRM. Overall we observed that UV-A radiation has a drastic effect on the photosynthetic machinery, photosynthetic pigments and intracellular amino acids. As a mitigation strategy against UV-A radiation, proline, glycine, and tyrosine were accumulated. Copyright © 2014. Published by Elsevier B.V.

  8. Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks

    Isabel G. Fernández de Mera

    2017-07-01

    Full Text Available Crimean-Congo hemorrhagic fever (CCHF is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV. In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.

  9. Large-scale proteomic identification of S100 proteins in breast cancer tissues

    Cancemi, Patrizia; Di Cara, Gianluca; Albanese, Nadia Ninfa; Costantini, Francesca; Marabeti, Maria Rita; Musso, Rosa; Lupo, Carmelo; Roz, Elena; Pucci-Minafra, Ida

    2010-01-01

    Attempts to reduce morbidity and mortality in breast cancer is based on efforts to identify novel biomarkers to support prognosis and therapeutic choices. The present study has focussed on S100 proteins as a potentially promising group of markers in cancer development and progression. One reason of interest in this family of proteins is because the majority of the S100 genes are clustered on a region of human chromosome 1q21 that is prone to genomic rearrangements. Moreover, there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast, and this is frequently associated with tumour progression. Samples of breast cancer tissues were obtained during surgical intervention, according to the bioethical recommendations, and cryo-preserved until used. Tissue extracts were submitted to proteomic preparations for 2D-IPG. Protein identification was performed by N-terminal sequencing and/or peptide mass finger printing. The majority of the detected S100 proteins were absent, or present at very low levels, in the non-tumoral tissues adjacent to the primary tumor. This finding strengthens the role of S100 proteins as putative biomarkers. The proteomic screening of 100 cryo-preserved breast cancer tissues showed that some proteins were ubiquitously expressed in almost all patients while others appeared more sporadic. Most, if not all, of the detected S100 members appeared reciprocally correlated. Finally, from the perspective of biomarkers establishment, a promising finding was the observation that patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. This article reports for the first time the comparative proteomic screening of several S100 protein members among a large group of breast cancer patients. The results obtained strongly support the hypothesis that a significant deregulation of multiple S100 protein members is

  10. Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers

    Ray, Sandipan; Renu, Durairaj; Srivastava, Rajneesh; Gollapalli, Kishore; Taur, Santosh; Jhaveri, Tulip; Dhali, Snigdha; Chennareddy, Srinivasarao; Potla, Ankit; Dikshit, Jyoti Bajpai; Srikanth, Rapole; Gogtay, Nithya; Thatte, Urmila; Patankar, Swati; Srivastava, Sanjeeva

    2012-01-01

    This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates

  11. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers.

    Sandipan Ray

    Full Text Available This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM (n = 20, vivax malaria (VM (n = 17 and healthy controls (HC (n = 20 were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC. Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05 serum proteins were identified in FM and VM respectively, and almost half (46.2% of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates

  12. Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

    Pham, T.K.; Sierocinski, P.; Oost, van der J.; Wright, P.C.

    2010-01-01

    A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30 % of the total

  13. Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

    Panga Jaipal Reddy

    Full Text Available Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

  14. Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

    Liu, Peng

    2015-02-27

    Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response.

  15. Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

    Duijvesz, Diederick; Burnum-Johnson, Kristin E.; Gritsenko, Marina A.; Hoogland, Marije; Vredenbregt-van den Berg, Mirella S.; Willemsen, Rob; Luider, Theo N.; Pasa-Tolic, Ljiljana; Jenster, Guido

    2013-12-31

    Introduction: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, biomarker discovery from body fluids is often hampered by the high abundance of many proteins unrelated to disease. An attractive alternative biomarker discovery approach is the isolation of small vesicles (exosomes, ~100 nm). They contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific marker discovery. Profiling prostate cancer-derived exosomes could reveal new markers for this malignancy. Materials and Methods: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. Proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode, followed by the Accurate Mass and Time (AMT) tag approach. Exosomal proteins were validated by Western blotting. A Tissue Micro Array, containing 481 different PCa samples (radical prostatectomy), was used to correlate candidate markers with several clinical-pathological parameters such as PSA, Gleason score, biochemical recurrence, and (PCa-related) death. Results: Proteomic characterization resulted in the identification of 263 proteins by at least 2 peptides. Specifically analysis of exosomes from PNT2C2, RWPE-1, PC346C, and VCaP identified 248, 233, 169, and 216 proteins, respectively. Statistical analyses revealed 52 proteins differently expressed between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. The Tissue Micro 4 Array showed strong correlation of higher Gleason scores and local recurrence with increased cytoplasmic XPO1 (P<0.001). Conclusions: Differentially abundant proteins of cell line-derived exosomes make a clear subdivision between

  16. The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

    Thomas Gronemeyer

    Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

  17. Automatic and rapid identification of glycopeptides by nano-UPLC-LTQ-FT-MS and proteomic search engine.

    Giménez, Estela; Gay, Marina; Vilaseca, Marta

    2017-01-30

    Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578. We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Urinary proteomic diagnosis of coronary artery disease: identification and clinical validation in 623 individuals

    Delles, Christian; Schiffer, Eric; von Zur Muhlen, Constantin

    2010-01-01

    We studied the urinary proteome in a total of 623 individuals with and without coronary artery disease (CAD) in order to characterize multiple biomarkers that enable prediction of the presence of CAD....

  19. Optimized method for identification of the proteomes secreted by cardiac cells

    Šťastná, Miroslava; Van Eyk, J.E.

    2013-01-01

    Roč. 1005, č. 1005 (2013), s. 225-235 ISSN 1940 -6029 Institutional support: RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

  20. Identification of β2-microglobulin as a urinary biomarker for chronic allograft nephropathy using proteomic methods.

    Johnston, Olwyn

    2011-08-01

    Chronic allograft nephropathy (CAN) remains the leading cause of renal graft loss after the first year following renal transplantation. This study aimed to identify novel urinary proteomic profiles, which could distinguish and predict CAN in susceptible individuals.

  1. Optimized method for identification of the proteomes secreted by cardiac cells

    Šťastná, Miroslava; Van Eyk, J.E.

    2013-01-01

    Roč. 1005, č. 1005 (2013), s. 225-235 ISSN 1940-6029 Institutional support: RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

  2. Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

    Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2014-01-01

    Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

  3. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  4. Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

    Sylvie Bourassa

    Full Text Available Insulin resistance (IR is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra. Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2 of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

  5. iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry

    Kambiranda, Devaiah; Katam, Ramesh; Basha, Sheikh M.; Siebert, Shalom

    2014-01-01

    Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening. PMID:24251720

  6. Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

    Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

    2015-11-01

    Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Proteomics of Pyrococcus furiosus (Pfu): Identification of Extracted Proteins by Three Independent Methods.

    Wong, Catherine C L; Cociorva, Daniel; Miller, Christine A; Schmidt, Alexander; Monell, Craig; Aebersold, Ruedi; Yates, John R

    2013-02-01

    Pyrococcus furiosus (Pfu) is an excellent organism to generate reference samples for proteomics laboratories because of its moderately sized genome and very little sequence duplication within the genome. We demonstrated a stable and consistent method to prepare proteins in bulk that eliminates growth and preparation as a source of uncertainty in the standard. We performed several proteomic studies in different laboratories using each laboratory's specific workflow as well as separate and integrated data analysis. This study demonstrated that a Pfu whole cell lysate provides suitable protein sample complexity to not only validate proteomic methods, work flows, and benchmark new instruments but also to facilitate comparison of experimental data generated over time and across instruments or laboratories.

  8. Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

    Šťastná, Miroslava; Chimenti, I.; Marban, E.; Van Eyk, J.E.

    2010-01-01

    Roč. 10, č. 2 (2010), s. 245-253 ISSN 1615-9853 Institutional research plan: CEZ:AV0Z40310501 Keywords : animal proteomics * cardiac stem cells * neonatal cardiomyocytes Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.815, year: 2010

  9. Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

    2014-01-01

    Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

  10. Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias).

    Lee, Jinoo; Valkova, Nelly; White, Mark P; Kültz, Dietmar

    2006-09-01

    We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.

  11. A proteomics approach to the identification of biomarkers for psoriasis utilising keratome biopsy

    Williamson, James C; Scheipers, Peter; Schwämmle, Veit

    2013-01-01

    a quantitative proteomics screen of four patients with psoriasis using stable isotope dimethyl labelling and identified over 50 proteins consistently altered in abundance in psoriasis lesional versus non-lesional skin. This includes several canonical psoriasis related proteins (e.g. S100A7 [Psoriasin] and FABP5...

  12. Maximizing the sensitivity and reliability of peptide identification in large-scale proteomic experiments by harnessing multiple search engines.

    Yu, Wen; Taylor, J Alex; Davis, Michael T; Bonilla, Leo E; Lee, Kimberly A; Auger, Paul L; Farnsworth, Chris C; Welcher, Andrew A; Patterson, Scott D

    2010-03-01

    Despite recent advances in qualitative proteomics, the automatic identification of peptides with optimal sensitivity and accuracy remains a difficult goal. To address this deficiency, a novel algorithm, Multiple Search Engines, Normalization and Consensus is described. The method employs six search engines and a re-scoring engine to search MS/MS spectra against protein and decoy sequences. After the peptide hits from each engine are normalized to error rates estimated from the decoy hits, peptide assignments are then deduced using a minimum consensus model. These assignments are produced in a series of progressively relaxed false-discovery rates, thus enabling a comprehensive interpretation of the data set. Additionally, the estimated false-discovery rate was found to have good concordance with the observed false-positive rate calculated from known identities. Benchmarking against standard proteins data sets (ISBv1, sPRG2006) and their published analysis, demonstrated that the Multiple Search Engines, Normalization and Consensus algorithm consistently achieved significantly higher sensitivity in peptide identifications, which led to increased or more robust protein identifications in all data sets compared with prior methods. The sensitivity and the false-positive rate of peptide identification exhibit an inverse-proportional and linear relationship with the number of participating search engines.

  13. Transcriptome and proteome analysis of Eucalyptus infected with Calonectria pseudoreteaudii.

    Chen, Quanzhu; Guo, Wenshuo; Feng, Lizhen; Ye, Xiaozhen; Xie, Wanfeng; Huang, Xiuping; Liu, Jinyan

    2015-02-06

    Cylindrocladium leaf blight is one of the most severe diseases in Eucalyptus plantations and nurseries. There are Eucalyptus cultivars with resistance to the disease. However, little is known about the defense mechanism of resistant cultivars. Here, we investigated the transcriptome and proteome of Eucalyptus leaves (E. urophylla×E. tereticornis M1), infected or not with Calonectria pseudoreteaudii. A total of 8585 differentially expressed genes (|log2 ratio| ≥1, FDR ≤0.001) at 12 and 24hours post-inoculation were detected using RNA-seq. Transcriptional changes for five genes were further confirmed by qRT-PCR. A total of 3680 proteins at the two time points were identified using iTRAQ technique.The combined transcriptome and proteome analysis revealed that the shikimate/phenylpropanoid pathway, terpenoid biosynthesis, signalling pathway (jasmonic acid and sugar) were activated. The data also showed that some proteins (WRKY33 and PR proteins) which have been reported to involve in plant defense response were up-regulated. However, photosynthesis, nucleic acid metabolism and protein metabolism were impaired by the infection of C. pseudoreteaudii. This work will facilitate the identification of defense related genes and provide insights into Eucalyptus defense responses to Cylindrocladium leaf blight. In this study, a total of 130 proteins and genes involved in the shikimate/phenylpropanoid pathway, terpenoid biosynthesis, signalling pathway, cell transport, carbohydrate and energy metabolism, nucleic acid metabolism and protein metabolism in Eucalyptus leaves after infected with C. pseudoreteaudii were identified. This is the first report of a comprehensive transcriptomic and proteomic analysis of Eucalyptus in response to Calonectria sp. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma.

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-04-09

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.

  15. Proteomic analysis of processing by-products from canned and fresh tuna: identification of potentially functional food proteins.

    Sanmartín, Esther; Arboleya, Juan Carlos; Iloro, Ibon; Escuredo, Kepa; Elortza, Felix; Moreno, F Javier

    2012-09-15

    Proteomic approaches have been used to identify the main proteins present in processing by-products generated by the canning tuna-industry, as well as in by-products derived from filleting of skeletal red muscle of fresh tuna. Following fractionation by using an ammonium sulphate precipitation method, three proteins (tropomyosin, haemoglobin and the stress-shock protein ubiquitin) were identified in the highly heterogeneous and heat-treated material discarded by the canning-industry. Additionally, this fractionation method was successful to obtain tropomyosin of high purity from the heterogeneous starting material. By-products from skeletal red muscle of fresh tuna were efficiently fractionated to sarcoplasmic and myofibrillar fractions, prior to the identification based mainly on the combined searching of the peptide mass fingerprint (MALDI-TOF) and peptide fragment fingerprinting (MALDI LIFT-TOF/TOF) spectra of fifteen bands separated by 1D SDS-PAGE. Thus, the sarcoplasmic fraction contained myoglobin and several enzymes that are essential for efficient energy production, whereas the myofibrillar fraction had important contractile proteins, such as actin, tropomyosin, myosin or an isoform of the enzyme creatine kinase. Application of proteomic technologies has revealed new knowledge on the composition of important by-products from tuna species, enabling a better evaluation of their potential applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest.

    Cologna, Nicholas de Mojana di; Gómez-Mendoza, Diana Paola; Zanoelo, Fabiana Fonseca; Giannesi, Giovana Cristina; Guimarães, Nelciele Cavalieri de Alencar; Moreira, Leonora Rios de Souza; Filho, Edivaldo Ximenes Ferreira; Ricart, Carlos André Ornelas

    2018-02-01

    Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Identification of 10 Candidate Biomarkers Distinguishing Tuberculous and Malignant Pleural Fluid by Proteomic Methods

    Lee, Chang Youl; Hong, Ji Young; Lee, Myung-Goo; Suh, In-Bum

    2017-01-01

    Purpose Pleural effusion, an accumulation of fluid in the pleural space, usually occurs in patients when the rate of fluid formation exceeds the rate of fluid removal. The differential diagnosis of tuberculous pleurisy and malignant pleural effusion is a difficult task in high tuberculous prevalence areas. The aim of the present study was to identify novel biomarkers for the diagnosis of pleural fluid using proteomics technology. Materials and Methods We used samples from five patients with t...

  18. Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid

    Lee Jae-Mok

    2011-07-01

    Full Text Available Abstract Background The inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS. Results We identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts. Conclusions Our results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.

  19. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid dep...... regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection....

  20. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

    2011-01-01

    Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  1. Identification of virulence determinants of the human pathogenic fungi Aspergillus fumigatus and Candida albicans by proteomics.

    Kniemeyer, Olaf; Schmidt, André D; Vödisch, Martin; Wartenberg, Dirk; Brakhage, Axel A

    2011-06-01

    Both fungi Candida albicans and Aspergillus fumigatus can cause a number of life-threatening systemic infections in humans. The commensal yeast C. albicans is one of the main causes of nosocomial fungal infectious diseases, whereas the filamentous fungus A. fumigatus has become one of the most prevalent airborne fungal pathogens. Early diagnosis of these fungal infections is challenging, only a limited number of antifungals for treatment are available, and the molecular details of pathogenicity are hardly understood. The completion of both the A. fumigatus and C. albicans genome sequence provides the opportunity to improve diagnosis, to define new drug targets, to understand the functions of many uncharacterised proteins, and to study protein regulation on a global scale. With the application of proteomic tools, particularly two-dimensional gel electrophoresis and LC/MS-based methods, a comprehensive overview about the proteins of A. fumigatus and C. albicans present or induced during environmental changes and stress conditions has been obtained in the past 5 years. However, for the discovery of further putative virulence determinants, more sensitive and targeted proteomic methods have to be applied. Here, we review the recent proteome data generated for A. fumigatus and C. albicans that are related to factors required for pathogenicity. Copyright © 2011 Elsevier GmbH. All rights reserved.

  2. Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders

    Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

    2017-01-01

    Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as wel...

  3. Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).

    Hammoudi, Abeer; Song, Fei; Reed, Karen R; Jenkins, Rosalind E; Meniel, Valerie S; Watson, Alastair J M; Pritchard, D Mark; Clarke, Alan R; Jenkins, John R

    2013-10-25

    Colorectal cancer (CRC) is the fourth most common cause of cancer-related death worldwide. Accurate non-invasive screening for CRC would greatly enhance a population's health. Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas, leading to Wnt signalling pathway activation. Acute conditional transgenic deletion of Apc in murine intestinal epithelium (AhCre(+)Apc(fl)(/)(fl)) causes phenotypic changes similar to those found during colorectal tumourigenesis. This study comprised a proteomic analysis of murine small intestinal epithelial cells following acute Apc deletion to identify proteins that show altered expression during human colorectal carcinogenesis, thus identifying proteins that may prove clinically useful as blood/serum biomarkers of colorectal neoplasia. Eighty-one proteins showed significantly increased expression following iTRAQ analysis, and validation of nine of these by Ingenuity Pathaway Analysis showed they could be detected in blood or serum. Expression was assessed in AhCre(+)Apc(fl)(/)(fl) small intestinal epithelium by immunohistochemistry, western blot and quantitative real-time PCR; increased nucelolin concentrations were also detected in the serum of AhCre(+)Apc(fl)(/)(fl) and Apc(Min)(/)(+) mice by ELISA. Six proteins; heat shock 60kDa protein 1, Nucleolin, Prohibitin, Cytokeratin 18, Ribosomal protein L6 and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5,were selected for further investigation. Increased expression of 4 of these was confirmed in human CRC by qPCR. In conclusion, several novel candidate biomarkers have been identified from analysis of transgenic mice in which the Apc gene was deleted in the intestinal epithelium that also showed increased expression in human CRC. Some of these warrant further investigation as potential serum-based biomarkers of human CRC. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Proteomics Core

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  5. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Yu-Hsuan Tu

    Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  6. iTRAQ based investigation of plasma proteins in HIV infected and HIV/HBV coinfected patients - C9 and KLK are related to HIV/HBV coinfection.

    Sun, Tao; Liu, Li; Wu, Ao; Zhang, Yujiao; Jia, Xiaofang; Yin, Lin; Lu, Hongzhou; Zhang, Lijun

    2017-10-01

    Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) share similar routes of transmission, and rapid progression of hepatic and immunodeficiency diseases has been observed in coinfected individuals. Our main objective was to investigate the molecular mechanism of HIV/HBV coinfections. We selected HIV infected and HIV/HBV coinfected patients with and without Highly Active Antiretroviral Therapy (HAART). Low abundance proteins enriched using a multiple affinity removal system (MARS) were labeled with isobaric tags for relative and absolute quantitation (iTRAQ) kits and analyzed using liquid chromatography-mass spectrometry (LC-MS). The differential proteins were analyzed by Gene Ontology (GO) database. A total of 41 differential proteins were found in HIV/HBV coinfected patients as compared to HIV mono-infected patients with or without HAART treatment, including 7 common HBV-regulated proteins. The proteins involved in complement and coagulation pathways were significantly enriched, including plasma kallikrein (KLK) and complement component C9 (C9). C9 and KLK were verified to be down-regulated in HIV/HBV coinfected patients through ELISA analysis. The present iTRAQ based proteomic analyses identified 7 proteins that are related to HIV/HBV coinfection. HBV might influence hepatic and immune functions by deregulating complement and coagulation pathways. C9 and KLK could potentially be used as targets for the treatment of HIV/HBV coinfections. Copyright © 2017. Published by Elsevier Ltd.

  7. Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers

    Renzoni Adriana

    2006-11-01

    Full Text Available Abstract Background To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. Results In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Conclusion Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations

  8. Identification of species- and tissue-specific proteins using proteomic strategy

    Chernukha, I. M.; Vostrikova, N. L.; Kovalev, L. I.; Shishkin, S. S.; Kovaleva, M. A.; Manukhin, Y. S.

    2017-09-01

    Proteomic technologies have proven to be very effective for detecting biochemical changes in meat products, such as changes in tissue- and species-specific proteins. In the tissues of cattle, pig, horse and camel M. longissimus dorsi both tissue- and species specific proteins were detected using two dimensional electrophoresis. Species-specific isoforms of several muscle proteins were also identified. The identified and described proteins of cattle, pig, horse and camel skeletal muscles (including mass spectra of the tryptic peptides) were added to the national free access database “Muscle organ proteomics”. This research has enabled the development of new highly sensitive technologies for meat product quality control against food fraud.

  9. Effect of iTRAQ Labeling on the Relative Abundance of Peptide Fragment Ions Produced by MALDI-MS/MS

    Gandhi, Tejas; Puri, Pranav; Fusetti, Fabrizia; Breitling, Rainer; Poolman, Bert; Permentier, Hjalmar P.

    The identification of proteins in proteomics experiments is usually based on mass information derived from tandem mass spectrometry data. To improve the performance of the identification algorithms, additional information available in the fragment peak intensity patterns has been shown to be useful.

  10. Application of Machine Learning to Proteomics Data: Classification and Biomarker Identification in Postgenomics Biology

    Swan, Anna Louise; Mobasheri, Ali; Allaway, David; Liddell, Susan

    2013-01-01

    Abstract Mass spectrometry is an analytical technique for the characterization of biological samples and is increasingly used in omics studies because of its targeted, nontargeted, and high throughput abilities. However, due to the large datasets generated, it requires informatics approaches such as machine learning techniques to analyze and interpret relevant data. Machine learning can be applied to MS-derived proteomics data in two ways. First, directly to mass spectral peaks and second, to proteins identified by sequence database searching, although relative protein quantification is required for the latter. Machine learning has been applied to mass spectrometry data from different biological disciplines, particularly for various cancers. The aims of such investigations have been to identify biomarkers and to aid in diagnosis, prognosis, and treatment of specific diseases. This review describes how machine learning has been applied to proteomics tandem mass spectrometry data. This includes how it can be used to identify proteins suitable for use as biomarkers of disease and for classification of samples into disease or treatment groups, which may be applicable for diagnostics. It also includes the challenges faced by such investigations, such as prediction of proteins present, protein quantification, planning for the use of machine learning, and small sample sizes. PMID:24116388

  11. Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis.

    Galdiero, Francesca; Bello, Anna Maria; Spina, Anna; Capiluongo, Anna; Liuu, Sophie; De Marco, Margot; Rosati, Alessandra; Capunzo, Mario; Napolitano, Maria; Vuttariello, Emilia; Monaco, Mario; Califano, Daniela; Turco, Maria Caterina; Chiappetta, Gennaro; Vinh, Joëlle; Chiappetta, Giovanni

    2018-01-30

    BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.

  12. iTRAQ-based Quantitative Proteomics Study in Patients with Refractory Mycoplasma pneumoniae Pneumonia.

    Yu, Jia-Lu; Song, Qi-Fang; Xie, Zhi-Wei; Jiang, Wen-Hui; Chen, Jia-Hui; Fan, Hui-Feng; Xie, Ya-Ping; Lu, Gen

    2017-09-25

    Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children and young adults. Although MP pneumonia is usually benign and self-limited, in some cases it can develop into life-threating refractory MP pneumonia (RMPP). However, the pathogenesis of RMPP is poorly understood. The identification and characterization of proteins related to RMPP could provide a proof of principle to facilitate appropriate diagnostic and therapeutic strategies for treating paients with MP. In this study, we used a quantitative proteomic technique (iTRAQ) to analyze MP-related proteins in serum samples from 5 patients with RMPP, 5 patients with non-refractory MP pneumonia (NRMPP), and 5 healthy children. Functional classification, sub-cellular localization, and protein interaction network analysis were carried out based on protein annotation through evolutionary relationship (PANTHER) and Cytoscape analysis. A total of 260 differentially expressed proteins were identified in the RMPP and NRMPP groups. Compared to the control group, the NRMPP and RMPP groups showed 134 (70 up-regulated and 64 down-regulated) and 126 (63 up-regulated and 63 down-regulated) differentially expressed proteins, respectively. The complex functional classification and protein interaction network of the identified proteins reflected the complex pathogenesis of RMPP. Our study provides the first comprehensive proteome map of RMPP-related proteins from MP pneumonia. These profiles may be useful as part of a diagnostic panel, and the identified proteins provide new insights into the pathological mechanisms underlying RMPP.

  13. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease

    Benjamin I Baarda

    2015-10-01

    Full Text Available Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight- threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s. Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines.

  14. Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

    Tsai, Meng-Tsz; Chen, Yu-Jen; Chen, Ching-Yi; Tsai, Mong-Hsun; Han, Chia-Li; Chen, Yu-Ju; Mersmann, Harry J; Ding, Shih-Torng

    2017-03-01

    Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model. Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis. Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 μM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined. Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S -transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition ( P steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro ( P < 0.05). Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic indicators for NAFLD in the future. © 2017 American Society for Nutrition.

  15. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    An, Xiaomeng; Shao, Jiaofang; Zhang, Huoming; Ren, Xiaoliang; Ho, Vincy Wing Sze; Li, Runsheng; Wong, Ming-Kin; Zhao, Zhongying

    2017-01-01

    . The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins

  16. A proteome study of secreted prostatic factors affecting osteoblastic activity, identification and characterisation of cyclophilin A

    Andersen, H.; Jensen, O.N.; Eriksen, E.F.

    2003-01-01

    ranging from 5 to 30 kDa were analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). One of these spots was identified as cyclophilin A. We examined whether cyclophilin A alone or in combination with insulin-like growth factor-I (IGF-I) had any effects...... was to characterise the protein profile of conditioned medium (CM) from PC3 cells in the molecular weight range of 5-30 kDa using proteome analysis. A protein profile of the CM from PC3 cells was performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Thirty protein spots with molecular weights...

  17. Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

    Fan Xiuzhen; Wang Jianling; Soman, Kizhake V.; Ansari, G.A.S.; Khan, M. Firoze

    2011-01-01

    Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6 kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity. - Highlights: → Proteomic approaches are used to identify nitrated proteins in the spleen. → Twenty five nitrated proteins were found only in the spleen of aniline-treated rats. → Aniline exposure led to increased iNOS mRNA and protein

  18. Identification of proteomic biomarkers of preeclampsia using protein microarray and tandem mass spectrometry

    Karol Charkiewicz

    2015-05-01

    Full Text Available Preeclampsia (PE is the leading cause of death of the fetus and the mother. The exact pathomechanism has not so far been clarified. PE coexists with many other diseases, but it is often difficult to explain the association between them and find a clear reason for their occurrence. There are many predictive factors, but none are highly specific in preeclampsia. The diagnosis of preeclampsia seems to be very complex, which is another argument for the exploration of knowledge on this subject. Although many of the discoveries have hitherto been made in the field of proteomics, still no single specific biomarker of preeclampsia has been discovered. Research at the genome level is important because it can help us understand the genetic predisposition of patients affected by this disease. Nevertheless, researchers have recently become more interested in the pathophysiology of PE, and they are trying to answer the question: what is the real, direct cause of preeclampsia? Thus, the discovery of a protein that is a good predictor of preeclampsia development would significantly accelerate the medical care of pregnant women, and consequently reduce the risk of occurrence of HELLP syndrome and fetal death. Apart from the predictive and diagnostic function, such a discovery would help us to better understand the pathogenesis of preeclampsia and to find in the future a medical drug to suppress this disease. In order to make a breakthrough in this field, scientists need to use the most modern methods of proteomics, which allow for the analysis of small amounts of biological material in the shortest possible time, thereby giving a lot of information about existing proteins in the sample. Such optimization allows two methods, most commonly used by researchers: tandem mass spectrometry and protein microarray technique.

  19. Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway.

    Lyle J Burdine

    Full Text Available Constitutive activation of the Rearranged during Transfection (RET proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC. The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.

  20. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Rosu-Myles, Michael; She, Yi-Min; Fair, Joel; Muradia, Gauri; Mehic, Jelica; Menendez, Pablo; Prasad, Shiv S; Cyr, Terry D

    2012-01-01

    Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  1. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  2. IdentiPy: an extensible search engine for protein identification in shotgun proteomics.

    Levitsky, Lev I; Ivanov, Mark V; Lobas, Anna A; Bubis, Julia A; Tarasova, Irina A; Solovyeva, Elizaveta M; Pridatchenko, Marina L; Gorshkov, Mikhail V

    2018-04-23

    We present an open-source, extensible search engine for shotgun proteomics. Implemented in Python programming language, IdentiPy shows competitive processing speed and sensitivity compared with the state-of-the-art search engines. It is equipped with a user-friendly web interface, IdentiPy Server, enabling the use of a single server installation accessed from multiple workstations. Using a simplified version of X!Tandem scoring algorithm and its novel ``auto-tune'' feature, IdentiPy outperforms the popular alternatives on high-resolution data sets. Auto-tune adjusts the search parameters for the particular data set, resulting in improved search efficiency and simplifying the user experience. IdentiPy with the auto-tune feature shows higher sensitivity compared with the evaluated search engines. IdentiPy Server has built-in post-processing and protein inference procedures and provides graphic visualization of the statistical properties of the data set and the search results. It is open-source and can be freely extended to use third-party scoring functions or processing algorithms, and allows customization of the search workflow for specialized applications.

  3. Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

    Shipitsin, M; Small, C; Choudhury, S; Giladi, E; Friedlander, S; Nardone, J; Hussain, S; Hurley, A D; Ernst, C; Huang, Y E; Chang, H; Nifong, T P; Rimm, D L; Dunyak, J; Loda, M; Berman, D M; Blume-Jensen, P

    2014-09-09

    Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

  4. Identification and proteomic analysis of a novel gossypol-degrading fungal strain.

    Yang, Xia; Sun, Jian-Yi; Guo, Jian-Lin; Weng, Xiao-Yan

    2012-03-15

    Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol-degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation. A fungal strain, AN-1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose-containing and gossypol-containing mycelium extracts. Two-dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0-66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The fungus AN-1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source. Copyright © 2011 Society of Chemical Industry.

  5. Identification of 10 Candidate Biomarkers Distinguishing Tuberculous and Malignant Pleural Fluid by Proteomic Methods.

    Lee, Chang Youl; Hong, Ji Young; Lee, Myung Goo; Suh, In Bum

    2017-11-01

    Pleural effusion, an accumulation of fluid in the pleural space, usually occurs in patients when the rate of fluid formation exceeds the rate of fluid removal. The differential diagnosis of tuberculous pleurisy and malignant pleural effusion is a difficult task in high tuberculous prevalence areas. The aim of the present study was to identify novel biomarkers for the diagnosis of pleural fluid using proteomics technology. We used samples from five patients with transudative pleural effusions for internal standard, five patients with tuberculous pleurisy, and the same numbers of patients having malignant effusions were enrolled in the study. We analyzed the proteins in pleural fluid from patients using a technique that combined two-dimensional liquid-phase electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. We identified a total of 10 proteins with statistical significance. Among 10 proteins, trasthyretin, haptoglobin, metastasis-associated protein 1, t-complex protein 1, and fibroblast growth factor-binding protein 1 were related with malignant pleural effusions and human ceruloplasmin, lysozyme precursor, gelsolin, clusterin C complement lysis inhibitor, and peroxirexdoxin 3 were expressed several times or more in tuberculous pleural effusions. Highly expressed proteins in malignant pleural effusion were associated with carcinogenesis and cell growth, and proteins associated with tuberculous pleural effusion played a role in the response to inflammation and fibrosis. These findings will aid in the development of novel diagnostic tools for tuberculous pleurisy and malignant pleural effusion of lung cancer. © Copyright: Yonsei University College of Medicine 2017

  6. Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis

    Kimura, Yayoi; Yanagimachi, Masakatsu; Ino, Yoko; Aketagawa, Mao; Matsuo, Michie; Okayama, Akiko; Shimizu, Hiroyuki; Oba, Kunihiro; Morioka, Ichiro; Imagawa, Tomoyuki; Kaneko, Tetsuji; Yokota, Shumpei; Hirano, Hisashi; Mori, Masaaki

    2017-01-01

    Kawasaki disease (KD) is a systemic vasculitis and childhood febrile disease that can lead to cardiovascular complications. The diagnosis of KD depends on its clinical features, and thus it is sometimes difficult to make a definitive diagnosis. In order to identify diagnostic serum biomarkers for KD, we explored serum KD-related proteins, which differentially expressed during the acute and recovery phases of two patients by mass spectrometry (MS). We identified a total of 1,879 proteins by MS-based proteomic analysis. The levels of three of these proteins, namely lipopolysaccharide-binding protein (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute phase patients. In contrast, the level of retinol-binding protein 4 (RBP4) was decreased. To confirm the usefulness of these proteins as biomarkers, we analyzed a total of 270 samples, including those collected from 55 patients with acute phase KD, by using western blot analysis and microarray enzyme-linked immunosorbent assays (ELISAs). Over the course of this experiment, we determined that the expression level of these proteins changes specifically in the acute phase of KD, rather than the recovery phase of KD or other febrile illness. Thus, LRG1 could be used as biomarkers to facilitate KD diagnosis based on clinical features. PMID:28262744

  7. Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

    Zong-Xiu Wang

    Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

  8. Organ-specific proteomics analysis for identification of response mechanism in soybean seedlings under flooding stress.

    Khatoon, Amana; Rehman, Shafiq; Hiraga, Susumu; Makino, Takahiro; Komatsu, Setsuko

    2012-10-22

    Flooding is one of the severe environmental factors which impair growth and yield in soybean plant. To investigate the organ specific response mechanism of soybean under flooding stress, changes in protein species were analyzed using a proteomics approach. Two-day-old soybeans were subjected to flooding for 5 days. Proteins were extracted from root, hypocotyl and leaf, and separated by two-dimensional polyacrylamide gel electrophoresis. In root, hypocotyl and leaf, 51, 66 and 51 protein species were significantly changed, respectively, under flooding stress. In root, metabolism related proteins were increased; however these proteins were decreased in hypocotyl and leaf. In all 3 organs, cytoplasm localized proteins were decreased, and leaf chloroplastic proteins were also decreased. Isoflavone reductase was commonly decreased at protein level in all 3 organs; however, mRNA of isoflavone reductase gene was up-regulated in leaf under flooding stress. Biophoton emission was increased in all 3 organs under flooding stress. The up-regulation of isoflavone reductase gene at transcript level; while decreased abundance at protein level indicated that flooding stress affected the mRNA translation to proteins. These results suggest that concurrence in expression of isoflavone reductase gene at mRNA and protein level along with imbalance in other disease/defense and metabolism related proteins might lead to impaired growth of root, hypocotyl and leaf of soybean seedlings under flooding stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network

    Cui, Jian; Liu, Jinghua; Li, Yuhua; Shi, Tieliu

    2011-01-01

    Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome. PMID:21297957

  10. Mass Spectrometric Identification and Differentiation of Botulinum Neurotoxins through Toxin Proteomics.

    Kalb, Suzanne R; Barr, John R

    2013-08-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence and immunogenic properties, and some subtypes are further differentiated into toxin variants. Toxin characterization is important as different types of BoNT can respond differently to medical countermeasures for botulism, and characterization of the toxin can aid in epidemiologic and forensic investigations. Proteomic techniques have been established to determine the serotype, subtype, or toxin variant of BoNT. These techniques involve digestion of the toxin into peptides, tandem mass spectrometric (MS/MS) analysis of the peptides, and database searching to identify the BoNT protein. These techniques demonstrate the capability to detect BoNT and its neurotoxin-associated proteins, and differentiate the toxin from other toxins which are up to 99.9% identical in some cases. This differentiation can be accomplished from toxins present in a complex matrix such as stool, food, or bacterial cultures and no DNA is required.

  11. Proteomic identification of an embryo-specific 1Cys-Prx promoter and analysis of its activity in transgenic rice.

    Kim, Je Hein; Jung, In Jung; Kim, Dool Yi; Fanata, Wahyu Indra; Son, Bo Hwa; Yoo, Jae Yong; Harmoko, Rikno; Ko, Ki Seong; Moon, Jeong Chan; Jang, Ho Hee; Kim, Woe Yeon; Kim, Jae-Yean; Lim, Chae Oh; Lee, Sang Yeol; Lee, Kyun Oh

    2011-04-29

    Proteomic analysis of a rice callus led to the identification of 10 abscisic acid (ABA)-induced proteins as putative products of the embryo-specific promoter candidates. 5'-flanking sequence of 1 Cys-Prx, a highly-induced protein gene, was cloned and analyzed. The transcription initiation site of 1 Cys-Prx maps 96 nucleotides upstream of the translation initiation codon and a TATA-box and putative seed-specific cis-acting elements, RYE and ABRE, are located 26, 115 and 124 bp upstream of the transcription site, respectively. β-glucuronidase (GUS) expression driven by the 1 Cys-Prx promoters was strong in the embryo and aleurone layer and the activity reached up to 24.9 ± 3.3 and 40.5 ± 2.1 pmol (4 MU/min/μg protein) in transgenic rice seeds and calluses, respectively. The activity of the 1 Cys-Prx promoters is much higher than that of the previously-identified embryo-specific promoters, and comparable to that of strong endosperm-specific promoters in rice. GUS expression driven by the 1 Cys-Prx promoters has been increased by ABA treatment and rapidly induced by wounding in callus and at the leaf of the transgenic plants, respectively. Furthermore, ectopic expression of the GUS construct in Arabidopsis suggested that the 1 Cys-Prx promoter also has strong activity in seeds of dicot plants. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Comparative study using phenotypic, genotypic and proteomics methods for identification of coagulase-negative staphylococci

    Dr. P.F.G. Wolffs; Ing M. Valkenburg; Dr. A.J.C. van den Brule, van den; M.Sc. A. Jansz; Drs A.J.M. Loonen; Ing J.N.B. Bergland

    2012-01-01

    Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be

  13. Quantitative proteomic analysis by iTRAQ® for the identification of candidate biomarkers in ovarian cancer serum

    Higgins LeeAnn

    2010-06-01

    Full Text Available Abstract Background Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ®. Results Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified. Conclusions This study provides the first analysis of immunodepleted serum in combination with iTRAQ® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

  14. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

    Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

    2010-05-01

    Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

  15. Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

    Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P

    2016-10-01

    Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Proteomic Identification of Non-Gal Antibody Targets After Pig-to-Primate Cardiac Xenotransplantation

    Byrne, Guerard W.; Stalboerger, Paul G.; Davila, Eduardo; Heppelmann, Carrie J.; Gazi, Mozammel H.; McGregor, Hugh C. J.; LaBreche, Peter T.; Davies, William R.; Rao, Vinay P.; Oi, Keiji; Tazelaar, Henry D.; Logan, John S.; McGregor, Christopher G. A.

    2008-01-01

    Background Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens. Methods To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by Western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses. PMID:18957049

  17. Proteomic identification of differentially expressed proteins during alfalfa (Medicago sativa L. flower development

    Lingling Chen

    2016-10-01

    Full Text Available Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L. seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1, pollination (S2, and the post-pollination senescence period (S3. Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD. Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs, carbonic anhydrase (CA, and NADPH: quinone oxidoreductase-like protein (NQOLs. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower

  18. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa ( Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  19. Identification of parvalbumin and two new thermolabile major allergens of Thunnus tonggol using a proteomics approach.

    Rosmilah, Misnan; Shahnaz, Murad; Meinir, Jones; Masita, Arip; Noormalin, Abdullah; Jamaluddin, Mohamed

    2013-01-01

    The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach. Raw and boiled extracts of the fish were prepared. Fish proteins were separated by means of SDS-PAGE and two-dimensional (2-DE) electrophoresis. 1-DE immunoblotting of raw extract was performed with sera from fish-allergic patients. Ten sera were further analysed by 2-DE immunoblotting. Selected major allergenic protein spots were excised, trypsin digested and analysed by means of mass spectrometry. SDS-PAGE of raw extract revealed 26 protein fractions, while boiled extract demonstrated fewer bands. The 2-DE gel profile of the raw extract further fractionated the protein bands to more than 100 distinct protein spots. 1-DE immunoblotting of raw extract exhibited two thermolabile protein fractions of 42 and 51 kDa as the major allergens, while the boiled extract only revealed a single IgE-binding band at 151 kDa. 2-DE immunoblotting of raw extract further detected numerous major IgE-reactive spots of 11-13, 42 and 51 kDa. Mass spectrometry analysis of the peptides generated from the 12, 42 and 51 kDa digested spots indicated that these spots were parvalbumin, creatine kinase and enolase, respectively. In addition to parvalbumin, two new thermolabile allergens were identified as major allergenic proteins of T. tonggol. This study proved that both thermostable and thermolabile proteins are important in local tuna allergy and should be included in diagnostic strategies.

  20. Proteomic identification of carbonylated proteins in F344 rat hippocampus after 1-bromopropane exposure

    Huang, Zhenlie [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466‐8550 (Japan); Department of Toxicology, Guangdong Prevention and Treatment Center for Occupational Diseases, Guangzhou 510‐300 (China); Ichihara, Sahoko [Graduate School of Regional Innovation Studies, Mie University, Tsu 514‐8507 (Japan); Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514‐8507 (Japan); Chang, Jie; Zhang, Lingyi; Subramanian, Kaviarasan; Mohideen, Sahabudeen Sheik [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466‐8550 (Japan); Ichihara, Gaku, E-mail: gak@med.nagoya-u.ac.jp [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466‐8550 (Japan)

    2012-08-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and humans. Previous proteomic analysis of rat hippocampus implicated alteration of protein expression in oxidative stress, suggesting that oxidative stress plays a role in 1-BP-induced neurotoxicity. To understand this role at the protein level, we exposed male F344 rats to 1-BP at 0, 400, or 1000 ppm for 8 h/day for 1 week or 4 weeks by inhalation and quantitated changes in hippocampal protein carbonyl using a protein carbonyl assay, two-dimensional gel electrophoresis (2-DE), immunoblotting, and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). Hippocampal reactive oxygen species and protein carbonyl were significantly increased, demonstrating 1-BP-associated induction of oxidative stress and protein damage. MALDI-TOF-TOF/MS identified 10 individual proteins with increased carbonyl modification (p < 0.05; fold-change ≥ 1.5). The identified proteins were involved in diverse biological processes including glycolysis, ATP production, tyrosine catabolism, GTP binding, guanine degradation, and neuronal metabolism of dopamine. Hippocampal triosephosphate isomerase (TPI) activity was significantly reduced and negatively correlated with TPI carbonylation (p < 0.001; r = 0.83). Advanced glycation end-product (AGE) levels were significantly elevated both in the hippocampus and plasma, and hippocampal AGEs correlated negatively with TPI activity (p < 0.001; r = 0.71). In conclusion, 1-BP-induced neurotoxicity in the rat hippocampus seems to involve oxidative damage of cellular proteins, decreased TPI activity, and elevated AGEs. -- Highlights: ► 1-BP increases hippocampal ROS levels and hippocampal and plasma protein carbonyls. ► 1-BP increases TPI carbonylation and decreases TPI activity in the hippocampus. ► 1-BP increases hippocampal and plasma AGE levels.

  1. From dysplastic nevus to melanoma: functional proteomic approach for the identification of bio markers

    De Pol, A.

    2009-01-01

    The project ultimately aims to identify bio markers from serum or other biological fluids helpful for early diagnosis of melanoma. Parametric analysis combined with advanced skin imaging technology, such as con focal microscopy, is directed to the identification of different types of benign melanocyte lesions, as well as to the characterization of different melanomas and dysplastic nevi, in order to understand different tumour progression behaviours and to identify possible melanoma precursors

  2. Proteomics-Based Identification of the Molecular Signatures of Liver Tissues from Aged Rats following Eight Weeks of Medium-Intensity Exercise

    Fanghui Li

    2016-01-01

    Full Text Available Physical activity has emerged as a powerful intervention that promotes healthy aging by maintaining the functional capacity of critical organ systems. Here, by combining functional and proteomics analyses, we examined how hepatic phenotypes might respond to exercise treatment in aged rats. 16 male aged (20 months old SD rats were divided into exercise and parallel control groups at random; the exercise group had 8 weeks of treadmill training with medium intensity. Whole protein samples of the liver were extracted from both groups and separated by two-dimensional gel electrophoresis. Alternatively objective protein spots with >2-fold difference in expression were selected for enzymological extraction and MS/MS identification. Results show increased activity of the manganese superoxide dismutase and elevated glutathione levels in the livers of exercise-treated animals, but malondialdehyde contents obviously decreased in the liver of the exercise group. Proteomics-based identification of differentially expressed proteins provided an integrated view of the metabolic adaptations occurring in the liver proteome during exercise, which significantly altered the expression of several proteins involved in key liver metabolic pathways including mitochondrial sulfur, glycolysis, methionine, and protein metabolism. These findings indicate that exercise may be beneficial to aged rats through modulation of hepatic protein expression profiles.

  3. Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis

    Shaimaa Abdelmegid

    2017-12-01

    Full Text Available Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

  4. Proteomic identification of differentially expressed proteins in aortic wall of patients with ruptured and nonruptured abdominal aortic aneurysms

    Urbonavicius, Sigitas; Lindholt, Jes S.; Vorum, Henrik

    2009-01-01

    To compare the basic proteomic composition of abdominal aortic aneurysm (AAA) wall tissue in patients with nonruptured and ruptured aneurysms.......To compare the basic proteomic composition of abdominal aortic aneurysm (AAA) wall tissue in patients with nonruptured and ruptured aneurysms....

  5. C4 photosynthetic machinery: insights from maize chloroplast proteomics

    Qi eZhao

    2013-04-01

    Full Text Available C4 plants exhibit much higher CO2 assimilation rates than C3 plants. The specialized differentiation of mesophyll cell (M and bundle sheath cell (BS type chloroplasts is unique to C4 plants and improves photosynthesis efficiency. Maize (Zea mays is an important crop and model with C4 photosynthetic machinery. Current high-throughput quantitative proteomics approaches (e.g., 2DE, iTRAQ, and shotgun proteomics have been employed to investigate maize chloroplast structure and function. These proteomic studies have provided valuable information on C4 chloroplast protein components, photosynthesis, and other metabolic mechanisms underlying chloroplast biogenesis, stromal and membrane differentiation, as well as response to salinity, high/low temperature, and light stress. This review presents an overview of proteomics advances in maize chloroplast biology.

  6. Differential Proteomics Identification of HSP90 as Potential Serum Biomarker in Hepatocellular Carcinoma by Two-dimensional Electrophoresis and Mass Spectrometry

    Yiyi Sun

    2010-03-01

    Full Text Available The aim of the current study is to identify the potential biomarkers involved in Hepatocellular carcinoma (HCC carcinogenesis. A comparative proteomics approach was utilized to identify the differentially expressed proteins in the serum of 10 HCC patients and 10 controls. A total of 12 significantly altered proteins were identified by mass spectrometry. Of the 12 proteins identified, HSP90 was one of the most significantly altered proteins and its over-expression in the serum of 20 HCC patients was confirmed using ELISA analysis. The observations suggest that HSP90 might be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of HCC. This work demonstrates that a comprehensive strategy of proteomic identification combined with further validation should be adopted in the field of cancer biomarker discovery.

  7. Identification of Hip BMD Loss and Fracture Risk Markers Through Population-Based Serum Proteomics: HIP BMD LOSS & FRACTURE RISK MARKERS BY POPULATION-BASED SERUM PROTEOMICS

    Nielson, Carrie; Wiedrick, Jack; Shen, Jian; Jacobs, Jon M.; Baker, Erin M.; Baraff, Aaron; Piehowski, Paul D.; Lee, Christine; Baratt, Arie; Petyuk, Vladislav A.; Mcweeney, Shannon K.; Lim, Jeong Youn; Bauer, Douglas C.; Lane, Nancy E.; Cawthon, Peggy M.; Smith, Richard D.; Lapidus, Jodi; Orwoll, Eric S.

    2017-04-06

    Accelerated bone loss significantly increases the risk of osteoporosis and fracture. The mechanisms underlying bone loss remain incompletely understood, and there are few available biomarkers. We utilized a novel proteomics approach to identify serum peptides and proteins associated with bone loss in 1967 older men who were randomly chosen from the Osteoporotic Fracture in Men Study (MrOS study) (age ≥ 65 yrs). Men had 2-3 measures of femoral neck BMD over an average follow-up of 4.6 years. Change in BMD was estimated and then categorized into three groups: maintained BMD (n=453), expected loss (n=1185) and accelerated loss (n=237). A liquid chromatography–ion mobility separation-mass spectrometry (LC-IMS-MS) proteomics platform was used to identify and quantify peptides from serum proteins. The whole cohort was randomly divided into discovery (N= 960) and validation (N= 915) sub-cohorts. Linear regression models and a random forest approach were used to discover differentially abundant individual peptides and a proteomic signature that distinguished individuals with accelerated bone loss from those who maintained BMD. Network analyses were performed using the MetaCore knowledgebase. We identified 12 peptides that were associated with BMD loss in both discovery (P< 0.1 FDR) and replication sub-cohorts (P<0.05). Those 12 peptides mapped to the following proteins: ALS, LYVE1, RNAS1, C2, ICOSL, C163A, C7, HEMO, CD14, CERU, CRAC1 and CD59. Meta-analysis of peptidesassociated with bone loss identified 6 additional proteins including GRP78, IGF-2, SHBG, ENPP2, IBP2 and IBP6. We also identified a proteomic signature that was predictive of BMD loss with a discriminative value similar to serum bone marker carboxy-terminal collagen crosslink peptide (CTX). Interestingly, combining the proteomic signature with CTX significantly improved the ability to discriminate men with accelerated loss. In summary, we have identified potential new biomarkers for bone loss that provide

  8. Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues

    Zhou, Jian-Ying; Chen, Lijun; Zhang, Bai; Tian, Yuan; Liu, Tao; Thomas, Stefani N.; Chen, Li; Schnaubelt, Michael; Boja, Emily; Hiltket, Tara; Kinsinger, Christopher; Rodriguez, Henry; Davies, Sherri; Li, Shunqiang; Snider, Jacqueline E.; Erdmann-Gilmore, Petra; Tabb, David L.; Townsend, Reid; Ellis, Matthew; Rodland, Karin D.; Smith, Richard D.; Carr, Steven A.; Zhang, Zhen; Chan, Daniel W.; Zhang, Hui

    2017-09-21

    The identification of protein biomarkers requires large-scale analysis of human specimens to achieve statistical significance. In this study, we evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification) based quantitative proteomics strategy using one channel for universal normalization across all samples. A total of 307 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating 107 one-dimensional (1D) LC-MS/MS datasets and 8 offline two-dimensional (2D) LC-MS/MS datasets (25 fractions for each set) for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we developed a quantification confidence score based on the quality of each peptide-spectrum match (PSM) to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS datasets collected over a 16 month period.

  9. iQuantitator: A tool for protein expression inference using iTRAQ

    Comte-Walters Susana

    2009-10-01

    Full Text Available Abstract Background Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™ [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions. Results This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report. Conclusion iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.

  10. Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

    Ruili Ma

    Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

  11. Plant iTRAQ-based proteomics

    Handakumbura, Pubudu; Hixson, Kim K.; Purvine, Samuel O.; Jansson, Georg C.; Pasa Tolic, Ljiljana

    2017-06-21

    We present a simple one-­pot extraction protocol, which rapidly isolates hydrophyllic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z-­score values, which normalize the expression data into a format that can easily be cross-­compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation.

  12. Proteomic identification of galectin-11 and 14 ligands from Haemonchus contortus

    Dhanasekaran Sakthivel

    2018-03-01

    Full Text Available Haemonchus contortus is the most pathogenic nematode of small ruminants. Infection in sheep and goats results in anaemia that decreases animal productivity and can ultimately cause death. The involvement of ruminant-specific galectin-11 (LGALS-11 and galectin-14 (LGALS-14 has been postulated to play important roles in protective immune responses against parasitic infection; however, their ligands are unknown. In the current study, LGALS-11 and LGALS-14 ligands in H. contortus were identified from larval (L4 and adult parasitic stages extracts using immobilised LGALS-11 and LGALS-14 affinity column chromatography and mass spectrometry. Both LGALS-11 and LGALS-14 bound more putative protein targets in the adult stage of H. contortus (43 proteins when compared to the larval stage (two proteins. Of the 43 proteins identified in the adult stage, 34 and 35 proteins were bound by LGALS-11 and LGALS-14, respectively, with 26 proteins binding to both galectins. Interestingly, hematophagous stage-specific sperm-coating protein and zinc metalloprotease (M13, which are known vaccine candidates, were identified as putative ligands of both LGALS-11 and LGALS-14. The identification of glycoproteins of H. contortus by LGALS-11 and LGALS-14 provide new insights into host-parasite interactions and the potential for developing new interventions.

  13. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approach

    Zupancic, Klemen; Blejec, Andrej; Herman, Ana; Veber, Matija; Verbovsek, Urska; Korsic, Marjan; Knezevic, Miomir; Rozman, Primoz; Turnsek, Tamara Lah; Gruden, Kristina; Motaln, Helena

    2014-01-01

    Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients. We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM. We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM. These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM. Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients

  14. Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

    George, Iniga S; Fennell, Anne Y; Haynes, Paul A

    2015-09-01

    Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System*

    Fritsch, Maximilian J.; Trunk, Katharina; Diniz, Juliana Alcoforado; Guo, Manman; Trost, Matthias; Coulthurst, Sarah J.

    2013-01-01

    It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial

  16. Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

    Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S

    2008-09-01

    The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

  17. Comparative Proteomics Analysis of Placenta from Pregnant Women with Intrahepatic Cholestasis of Pregnancy

    Zhang, Ting; Guo, Yueshuai; Guo, Xuejiang; Zhou, Tao; Chen, Daozhen; Xiang, Jingying; Zhou, Zuomin

    2013-01-01

    INTRODUCTION: Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach. METHODS: The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (...

  18. iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

    Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

    2017-01-30

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

  19. Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins.

    Banfi, Cristina; Baetta, Roberta; Gianazza, Erica; Tremoli, Elena

    2017-06-01

    Proteomic-based techniques provide a powerful tool for identifying the full spectrum of protein targets of a drug, elucidating its mechanism(s) of action, and identifying biomarkers of its efficacy and safety. Herein, we outline the technological advancements in the field, and illustrate the contribution of proteomics to the definition of the pharmacological profile of statins, which represent the cornerstone of the prevention and treatment of cardiovascular diseases (CVDs). Statins act by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, thus reducing cholesterol biosynthesis and consequently enhancing the clearance of low-density lipoproteins from the blood; however, HMG-CoA reductase inhibition can result in a multitude of additional effects beyond lipid lowering, known as 'pleiotropic effects'. The case of statins highlights the unique contribution of proteomics to the target profiling of a drug molecule. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Proteomics in medical microbiology.

    Cash, P

    2000-04-01

    The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

  1. Quantitative iTRAQ-Based Proteomic Identification of Candidate Biomarkers for Diabetic Nephropathy in Plasma of Type 1 Diabetic Patients

    Overgaard, Anne Julie; Thingholm, Tine Engberg; Larsen, Martin R

    2010-01-01

    INTRODUCTION: As part of a clinical proteomics programme focused on diabetes and its complications, it was our goal to investigate the proteome of plasma in order to find improved candidate biomarkers to predict diabetic nephropathy. METHODS: Proteins derived from plasma from a cross-sectional co...... nephropathy; however, they need to be confirmed in a longitudinal cohort. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9053-0) contains supplementary material, which is available to authorized users....

  2. Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

    Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

    2017-04-01

    Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  3. Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol.

    Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2013-01-14

    Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy.

    An, Mingrui; Lohse, Ines; Tan, Zhijing; Zhu, Jianhui; Wu, Jing; Kurapati, Himabindu; Morgan, Meredith A; Lawrence, Theodore S; Cuneo, Kyle C; Lubman, David M

    2017-04-07

    Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

  5. iTRAQ-based proteomics of sunflower cultivars differing in resistance to parasitic weed Orobanche cumana.

    Yang, Chong; Xu, Ling; Zhang, Na; Islam, Faisal; Song, Wenjian; Hu, Luyang; Liu, Dan; Xie, Xiaonan; Zhou, Weijun

    2017-07-01

    Orobanche cumana is an obligate root parasite causing severe damage to many economically important crops, including sunflowers worldwide. For efficient control measures, it is necessary to understand the resistant mechanism during interaction at molecular level. The present study emphasizes on comparative proteomics to investigate the mechanistic basis of compatible and incompatible interaction of O. cumana with resistant (JY207) and susceptible (TK0409) sunflowers. More than 3500 proteins were identified from two cultivars by iTRAQ analysis. Identified proteins associated with general functions, posttranslational modification, energy production and conversion, carbohydrate transport and metabolism, and signal transduction mechanisms were the most represented category of induced proteins in both cultivars. The resistant interaction was characterized by alteration of defense-related proteins involved in recognition of parasites, accumulation of pathogenesis-related proteins, biosynthesis of lignin, and detoxification of toxic metabolites in JY207 after inoculation. The susceptible interaction was characterized by decreased abundance of proteins involved in biosynthesis and signaling of plant growth regulators including auxin, gibberellin, brassinosteroid, and ethylene in TK0409 after inoculation. The present study provides comprehensive details of proteins and differential modulation of pathways regulated under compatible and incompatible interaction, allowing the identification of important molecular components for development of sustainable resistance against this parasite. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

    Trinh, Hung V.; Grossmann, Jonas; Gehrig, Peter; Roschitzki, Bernd; Schlapbach, Ralph; Greber, Urs F.; Hemmi, Silvio

    2013-01-01

    Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or lab...

  7. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  8. Optimization of mass spectrometric parameters improve the identification performance of capillary zone electrophoresis for single-shot bottom-up proteomics analysis.

    Zhang, Zhenbin; Dovichi, Norman J

    2018-02-25

    The effects of MS1 injection time, MS2 injection time, dynamic exclusion time, intensity threshold, and isolation width were investigated on the numbers of peptide and protein identifications for single-shot bottom-up proteomics analysis using CZE-MS/MS analysis of a Xenopus laevis tryptic digest. An electrokinetically pumped nanospray interface was used to couple a linear-polyacrylamide coated capillary to a Q Exactive HF mass spectrometer. A sensitive method that used a 1.4 Th isolation width, 60,000 MS2 resolution, 110 ms MS2 injection time, and a top 7 fragmentation produced the largest number of identifications when the CZE loading amount was less than 100 ng. A programmable autogain control method (pAGC) that used a 1.4 Th isolation width, 15,000 MS2 resolution, 110 ms MS2 injection time, and top 10 fragmentation produced the largest number of identifications for CZE loading amounts greater than 100 ng; 7218 unique peptides and 1653 protein groups were identified from 200 ng by using the pAGC method. The effect of mass spectrometer conditions on the performance of UPLC-MS/MS was also investigated. A fast method that used a 1.4 Th isolation width, 30,000 MS2 resolution, 45 ms MS2 injection time, and top 12 fragmentation produced the largest number of identifications for 200 ng UPLC loading amount (6025 unique peptides and 1501 protein groups). This is the first report where the identification number for CZE surpasses that of the UPLC at the 200 ng loading level. However, more peptides (11476) and protein groups (2378) were identified by using UPLC-MS/MS when the sample loading amount was increased to 2 μg with the fast method. To exploit the fast scan speed of the Q-Exactive HF mass spectrometer, higher sample loading amounts are required for single-shot bottom-up proteomics analysis using CZE-MS/MS. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Proteomic characterisation of bovine and avian purified protein derivatives and identification of specific antigens for serodiagnosis of bovine tuberculosis

    Infantes-Lorenzo, José Antonio; Moreno, Inmaculada; Risalde, María de los Ángeles; Roy, Álvaro; Villar, Margarita; Romero, Beatriz; Ibarrola, Nieves; de la Fuente, José; Puentes, Eugenia; de Juan, Lucía; Gortázar, Christian; Bezos, Javier; Domínguez, Lucas; Domínguez, Mercedes

    2017-01-01

    Background Bovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD) are widely used for bovine tuberculosis diagnosis. However, little is known about their qualitative and quantitative characteristics, which makes their standardisation difficult. In addition, bPPD can give false-positive tuberculosis results because of sequence homology between Mycobacterium bovis (M. bovis) and M. avium proteins. Thus, the objective of this study was to carry out a proteomic cha...

  10. Proteomic and transcriptomic profiling of in vitro established radiation resistant oral cancer cells for identification of radioresistance related biomarkers

    Mohd Yasser; Pawar, Sagar; Teni, Tanuja

    2016-01-01

    Radiotherapy is an integral part of oral cancer treatment, either alone or in combination with surgery. But, during radiotherapy, oral tumours of a subset of patients develop radioresistance that creates major obstruction towards its efficacy. The aim of our study was to establish radioresistant cell lines from different oral subsites using clinically admissible low dose radiation and profile them by proteomic and transcriptomic approaches to identify proteins associated with radioresistance in oral cancer

  11. Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics

    Dong, Wen; Kieliszewski, Marcia; Held, Michael A.

    2014-01-01

    The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolublization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we’ve identified tomato EP candidates using both proteomic and b...

  12. Bacterial membrane proteomics.

    Poetsch, Ansgar; Wolters, Dirk

    2008-10-01

    About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

  13. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

    2009-01-01

    Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

  14. iTRAQ Protein Profile Differential Analysis of Dormant and Germinated Grassbur Twin Seeds Reveals that Ribosomal Synthesis and Carbohydrate Metabolism Promote Germination Possibly Through the PI3K Pathway.

    Zhang, Guo-Liang; Zhu, Yue; Fu, Wei-Dong; Wang, Peng; Zhang, Rui-Hai; Zhang, Yan-Lei; Song, Zhen; Xia, Gui-Xian; Wu, Jia-He

    2016-06-01

    Grassbur is a destructive and invasive weed in pastures, and its burs can cause gastric damage to animals. The strong adaptability and reproductive potential of grassbur are partly due to a unique germination mechanism whereby twin seeds develop in a single bur: one seed germinates, but the other remains dormant. To investigate the molecular mechanism of seed germination in twin seeds, we used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a dynamic proteomic analysis of germination and dormancy. A total of 1,984 proteins were identified, 161 of which were considered to be differentially accumulated. The differentially accumulated proteins comprised 102 up-regulated and 59 down-regulated proteins. These proteins were grouped into seven functional categories, ribosomal proteins being the predominant group. The authenticity and accuracy of the results were confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription-PCR (qPCR). A dynamic proteomic analysis revealed that ribosome synthesis and carbohydrate metabolism affect seed germination possibly through the phosphoinositide 3-kinase (PI3K) pathway. As the PI3K pathway is generally activated by insulin, analyses of seeds treated with exogenous insulin by qPCR, ELISA and iTRAQ confirmed that the PI3K pathway can be activated, which suppresses dormancy and promotes germination in twin grassbur seeds. Together, these results show that the PI3K pathway may play roles in stimulating seed germination in grassbur by modulating ribosomal synthesis and carbohydrate metabolism. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography-tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p.

    Bailey, Ulla-Maja; Schulz, Benjamin L

    2013-04-01

    Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation sites. Optimal identification across the proteome was achieved with PNGase F deglycosylation and complementary proteolysis with either AspN or trypsin. We used this combination of deglycosylation and complementary protease digest to identify changes in the yeast cell wall proteome caused by lack of the Alg3p protein, a key component of the biosynthetic pathway of protein N-glycosylation. The cell wall of yeast lacking Alg3p showed specifically increased levels of Cis3p, a protein important for cell wall integrity. Our results showed that deglycosylation prior to protease digestion improved the quality of proteomic analyses even if protein glycosylation is not of direct relevance to the study at hand. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis.

    Mokada-Gopal, Lavanya; Boeser, Alexander; Lehmann, Christian H K; Drepper, Friedel; Dudziak, Diana; Warscheid, Bettina; Voehringer, David

    2017-05-01

    The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated >3-fold by IL-4 in a STAT6-dependent manner. Across all samples, ∼5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed >3-fold between IL-4-stimulated wild-type and STAT6 -/- B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5Rα, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor-like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4-regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity. Copyright © 2017 by The American Association of Immunologists, Inc.

  17. Proteomics analyses of human optic nerve head astrocytes following biomechanical strain.

    Rogers, Ronan S; Dharsee, Moyez; Ackloo, Suzanne; Sivak, Jeremy M; Flanagan, John G

    2012-02-01

    We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.

  18. Proteomics Analyses of Human Optic Nerve Head Astrocytes Following Biomechanical Strain*

    Rogers, Ronan S.; Dharsee, Moyez; Ackloo, Suzanne; Sivak, Jeremy M.; Flanagan, John G.

    2012-01-01

    We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin. PMID:22126795

  19. Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

    Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

    2013-11-01

    Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean.

  20. Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics.

    Dong, Wen; Kieliszewski, Marcia; Held, Michael A

    2015-04-01

    The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated (CG5). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in Escherichia coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach.

    Acosta-Muñiz, Carlos H; Escobar-Tovar, Lina; Valdes-Rodríguez, Silvia; Fernández-Pavia, Silvia; Arias-Saucedo, Luis J; de la Cruz Espindola Barquera, Maria; Gómez Lim, Miguel Á

    2012-01-01

    Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques. Copyright © Physiologia Plantarum 2011.

  2. Clinical proteomics

    Albrethsen, Jakob; Frederiksen, Hanne; Johannsen, Trine Holm

    2018-01-01

    Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS)-platforms...... standards and calibrants. The present challenge is to examine if targeted proteomics of IGF-I can truly measure up to the routine performance that must be expected from a clinical testing platform.......Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS......)-platforms already implemented in many clinical laboratories for routine quantitation of small molecules (i.e. uHPLC coupled to triple-quadrupole MS). Progress in targeted proteomics of circulating insulin-like growth factor 1 (IGF-I) have provided valuable insights about tryptic peptides, transitions, internal...

  3. Proteomics dataset

    Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2017-01-01

    The datasets presented in this article are related to the research articles entitled “Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies” (Bennike et al., 2015 [1]), and “Proteome Analysis of Rheumatoid Arthritis Gut Mucosa” (Bennike et al., 2017 [2])...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

  4. Proteomic analysis of Pinus radiata needles: 2-DE map and protein identification by LC/MS/MS and substitution-tolerant database searching.

    Valledor, Luis; Castillejo, Maria A; Lenz, Christof; Rodríguez, Roberto; Cañal, Maria J; Jorrín, Jesús

    2008-07-01

    Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

  5. Proteomics Insights into Autophagy.

    Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

    2017-10-01

    Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources

    Sunil S. Adav; Anita Ravindran; Siu Kwan Sze

    2015-01-01

    Here, we provide data related to the research article entitled ?Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes? by Adav et al. (J. Proteomics (2015) [1]). Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using i...

  7. Identification of targets of miR-200b by a SILAC-based quantitative proteomic approach

    Arivusudar Marimuthu

    2014-09-01

    Full Text Available miRNAs regulate gene expression by binding to cognate mRNAs causing mRNA degradation or translational repression. Mass spectrometry-based proteomic analysis is being widely used to identify miRNA targets. The miR-200b miRNA cluster is often overexpressed in multiple cancer types, but the identity of the targets remains elusive. Using SILAC-based analysis, we examined the effects of overexpression of a miR-200b mimic or a control miRNA in fibrosarcoma cells. We identified around 300 potential targets of miR-200b based on a change in the expression of protein levels. We validated a subset of potential targets at the transcript level using quantitative PCR.

  8. Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis.

    Cristina Herrera

    2016-04-01

    Full Text Available The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM and other extracellular matrix (ECM proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.

  9. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  10. Mining the human urine proteome for monitoring renal transplant injury

    Sigdel, Tara K.; Gao, Yuqian; He, Jintang; Wang, Anyou; Nicora, Carrie D.; Fillmore, Thomas L.; Shi, Tujin; Webb-Robertson, Bobbie-Jo; Smith, Richard D.; Qian, Wei-Jun; Salvatierra, Oscar; Camp, David G.; Sarwal, Minnie M.

    2016-06-01

    The human urinary proteome reflects systemic and inherent renal injury perturbations and can be analyzed to harness specific biomarkers for different kidney transplant injury states. 396 unique urine samples were collected contemporaneously with an allograft biopsy from 396 unique kidney transplant recipients. Centralized, blinded histology on the graft was used to classify matched urine samples into categories of acute rejection (AR), chronic allograft nephropathy (CAN), BK virus nephritis (BKVN), and stable graft (STA). Liquid chromatography–mass spectrometry (LC-MS) based proteomics using iTRAQ based discovery (n=108) and global label-free LC-MS analyses of individual samples (n=137) for quantitative proteome assessment were used in the discovery step. Selected reaction monitoring (SRM) was applied to identify and validate minimal urine protein/peptide biomarkers to accurately segregate organ injury causation and pathology on unique urine samples (n=151). A total of 958 proteins were initially quantified by iTRAQ, 87% of which were also identified among 1574 urine proteins detected in LC-MS validation. 103 urine proteins were significantly (p<0.05) perturbed in injury and enriched for humoral immunity, complement activation, and lymphocyte trafficking. A set of 131 peptides corresponding to 78 proteins were assessed by SRM for their significance in an independent sample cohort. A minimal set of 35 peptides mapping to 33 proteins, were modeled to segregate different injury groups (AUC =93% for AR, 99% for CAN, 83% for BKVN). Urinary proteome discovery and targeted validation identified urine protein fingerprints for non-invasive differentiation of kidney transplant injuries, thus opening the door for personalized immune risk assessment and therapy.

  11. Diet-induced perturbation of the rat liver mitochondrial acetylome studied by quantitative (iTRAQ) LC-MS/MS

    León, Ileana R.; Schwämmle, Veit; Williamson, James

    described3. Quantitation by MS/MS was achieved by using iTRAQ 8-Plex reagents (Life Technologies). LC-MS/MS analyses were performed using an Easy-nLC (Proxeon/ThermoFisher) fitted with an in-house made 17 cm C18 column that was interfaced to an LTQ-OrbiTrap XL instrument (ThermoFisher). Data analysis...

  12. Respiratory Proteomics Today: Are Technological Advances for the Identification of Biomarker Signatures Catching up with Their Promise? A Critical Review of the Literature in the Decade 2004-2013.

    Viglio, Simona; Stolk, Jan; Iadarola, Paolo; Giuliano, Serena; Luisetti, Maurizio; Salvini, Roberta; Fumagalli, Marco; Bardoni, Anna

    2014-01-22

    To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

  13. Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

    Lagrain, Bert; Brunnbauer, Markus; Rombouts, Ine; Koehler, Peter

    2013-01-01

    The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for

  14. Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

    Bert Lagrain

    Full Text Available The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS, the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC, and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%, the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and

  15. Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation.

    Zhou, Chun-Xue; Zhu, Xing-Quan; Elsheikha, Hany M; He, Shuai; Li, Qian; Zhou, Dong-Hui; Suo, Xun

    2016-10-04

    Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention. The development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified

  16. Identification of altered plasma proteins by proteomic study in valvular heart diseases and the potential clinical significance.

    Ge Gao

    Full Text Available BACKGROUND: Little is known about genetic basis and proteomics in valvular heart disease (VHD including rheumatic (RVD and degenerative (DVD valvular disease. The present proteomic study examined the hypothesis that certain proteins may be associated with the pathological changes in the plasma of VHD patients. METHODS AND RESULTS: Differential protein analysis in the plasma identified 18 differentially expressed protein spots and 14 corresponding proteins or polypeptides by two-dimensional electrophoresis and mass spectrometry in 120 subjects. Two up-regulated (complement C4A and carbonic anhydrase 1 and three down-regulated proteins (serotransferrin, alpha-1-antichymotrypsin, and vitronectin were validated by ELISA in enlarging samples. The plasma levels (n = 40 for each of complement C4A in RVD (715.8±35.6 vs. 594.7±28.2 ng/ml, P = 0.009 and carbonic anhydrase 1 (237.70±15.7 vs. 184.7±10.8 U/L, P = 0.007 in DVD patients were significantly higher and that of serotransferrin (2.36±0.20 vs. 2.93±0.16 mg/ml, P = 0.025 and alpha-1-antichymotrypsin (370.0±13.7 vs. 413.0±11.6 µg/ml, P = 0.019 in RVD patients were significantly lower than those in controls. The plasma vitronectin level in both RVD (281.3±11.0 vs. 323.2±10.0 µg/ml, P = 0.006 and DVD (283.6±11.4 vs. 323.2±10.0 µg/ml, P = 0.011 was significantly lower than those in normal controls. CONCLUSIONS: We have for the first time identified alterations of 14 differential proteins or polypeptides in the plasma of patients with various VHD. The elevation of plasma complement C4A in RVD and carbonic anhydrase 1 in DVD and the decrease of serotransferrin and alpha-1-antichymotrypsin in RVD patients may be useful biomarkers for these valvular diseases. The decreased plasma level of vitronectin - a protein related to the formation of valvular structure - in both RVD and DVD patients might indicate the possible genetic deficiency in these patients.

  17. Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin ▿ †

    Cagas, Steven E.; Jain, Mohit Raja; Li, Hong; Perlin, David S.

    2011-01-01

    The proteomic response of Aspergillus fumigatus to caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 μg/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy. PMID:20974863

  18. A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers

    Vasiliki Taraslia

    2018-01-01

    Full Text Available Dental stem cells (DSCs have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs and Periodontal Ligament Stem Cells (PDLSCs. A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

  19. Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

    Sung, Jin-Hee; Cho, Eun-Hae; Cho, Jae-Hyeon; Won, Chung-Kil; Kim, Myeong-Ok; Koh, Phil-Ok

    2012-11-01

    Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

  20. Identification of EBP50 as a Specific Biomarker for Carcinogens Via the Analysis of Mouse Lymphoma Cellular Proteome

    Lee, Yoen Jung; Choi, In-Kwon; Sheen, Yhun Yhong; Park, Sue Nie; Kwon, Ho Jeong

    2012-01-01

    To identify specific biomarkers generated upon exposure of L5178Y mouse lymphoma cells to carcinogens, 2-DE and MALDI-TOF MS analysis were conducted using the cellular proteome of L5178Y cells that had been treated with the known carcinogens, 1,2-dibromoethane and O-nitrotoluene and the noncarcinogens, emodin and D-mannitol. Eight protein spots that showed a greater than 1.5-fold increase or decrease in intensity following carcinogen treatment compared with treatment with noncarcinogens were selected. Of the identified proteins, we focused on the candidate biomarker ERM-binding phosphoprotein 50 (EBP50), the expression of which was specifically increased in response to treatment with the carcinogens. The expression level of EBP50 was determined by western analysis using polyclonal rabbit anti-EBP50 antibody. Further, the expression level of EBP50 was increased in cells treated with seven additional carcinogens, verifying that EBP50 could serve as a specific biomarker for carcinogens. PMID:22434383

  1. Proteomics approaches for identification of tumor relevant protein targets in pulmonary squamous cell carcinoma by 2D-DIGE-MS.

    Hao Lihong

    Full Text Available Potential markers for progression of pulmonary squamous cell carcinoma (SCC were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF. The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P ≤ 0.05 in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.

  2. RADARS, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database.

    Field, Helen I; Fenyö, David; Beavis, Ronald C

    2002-01-01

    RADARS, a rapid, automated, data archiving and retrieval software system for high-throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close-to-expert accuracy and brings robust data analysis to the non-expert user.

  3. Proteomic characterization of Aspergillus fumigatus treated with an antifungal coumarin for identification of novel target molecules of key pathways.

    Singh, Seema; Gupta, Shilpi; Singh, Bharat; Sharma, Sunil K; Gupta, Vijay K; Sharma, Gainda L

    2012-06-01

    A synthetic coumarin, N,N,N-triethyl-11-(4-methyl-2-oxo-2H-chromen-7-yloxy)-11-oxoundecan-1-aminium bromide (SCD-1), having potent activity against pathogenic Aspergilli (MIC90 15.62 μg/mL), was investigated to identify its molecular targets in the pathogen. The proteome of Aspergillus fumigatus was developed after treatment with sublethal doses of compound and analyzed. The results demonstrated 143 differentially expressed proteins on treatment with SCD-1. The expression of four proteins, namely cell division control protein, ubiquitin-like activating enzyme, vacuolar ATP synthase catalytic subunit A, and UTP-glucose-1-phosphate uridylyltransferase of A. fumigatus, was completely inhibited, whereas there were 13 newly expressed and 96 overexpressed proteins, mainly belonging to stress pathway. The treatment of A. fumigatus with SCD-1 also led to attenuation of proteins involved in cell replication and other important biosynthetic processes, including riboflavin biosynthesis, which has been pathogen-specific. In addition to key enzymatic players and antioxidants, nine hypothetical proteins were also identified, seven of which have been novel, being described for the first time. As no cellular functions have yet been described for these hypothetical proteins, their alteration in response to SCD-1 provides significant information about their putative roles in pathogen defense.

  4. Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

    Qiao, Jie; Du, Zhiheng; Zhang, Yueling; Du, Hong; Guo, Lingling; Zhong, Mingqi; Cao, Jingsong; Wang, Xiuying

    2011-12-01

    Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

    Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

    2013-07-01

    This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

  6. Identification of new serum markers of pathological states by bioinformatic tools for the analysis of serum proteomics expression profiles

    Malorni, A.; Facchiano, A.

    2009-01-01

    We have developed new bioinformatic tools and strategies, aimed to the identification and characterization of proteins as markers of pathological states, for the analysis of data derived from protein expression profiles obtained by mass spectrometry techniques, for the study of structural and functional properties of the proteins, and for the analysis of data from omics approaches

  7. Genomes to Proteomes

    Panisko, Ellen A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Grigoriev, Igor [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Daly, Don S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Webb-Robertson, Bobbie-Jo [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2009-03-01

    Biologists are awash with genomic sequence data. In large part, this is due to the rapid acceleration in the generation of DNA sequence that occurred as public and private research institutes raced to sequence the human genome. In parallel with the large human genome effort, mostly smaller genomes of other important model organisms were sequenced. Projects following on these initial efforts have made use of technological advances and the DNA sequencing infrastructure that was built for the human and other organism genome projects. As a result, the genome sequences of many organisms are available in high quality draft form. While in many ways this is good news, there are limitations to the biological insights that can be gleaned from DNA sequences alone; genome sequences offer only a bird's eye view of the biological processes endemic to an organism or community. Fortunately, the genome sequences now being produced at such a high rate can serve as the foundation for other global experimental platforms such as proteomics. Proteomic methods offer a snapshot of the proteins present at a point in time for a given biological sample. Current global proteomics methods combine enzymatic digestion, separations, mass spectrometry and database searching for peptide identification. One key aspect of proteomics is the prediction of peptide sequences from mass spectrometry data. Global proteomic analysis uses computational matching of experimental mass spectra with predicted spectra based on databases of gene models that are often generated computationally. Thus, the quality of gene models predicted from a genome sequence is crucial in the generation of high quality peptide identifications. Once peptides are identified they can be assigned to their parent protein. Proteins identified as expressed in a given experiment are most useful when compared to other expressed proteins in a larger biological context or biochemical pathway. In this chapter we will discuss the automatic

  8. Analysis of mass spectrometry data in proteomics

    Matthiesen, Rune; Jensen, Ole N

    2008-01-01

    The systematic study of proteins and protein networks, that is, proteomics, calls for qualitative and quantitative analysis of proteins and peptides. Mass spectrometry (MS) is a key analytical technology in current proteomics and modern mass spectrometers generate large amounts of high-quality data...... that in turn allow protein identification, annotation of secondary modifications, and determination of the absolute or relative abundance of individual proteins. Advances in mass spectrometry-driven proteomics rely on robust bioinformatics tools that enable large-scale data analysis. This chapter describes...... some of the basic concepts and current approaches to the analysis of MS and MS/MS data in proteomics....

  9. Identification of Changes in Wheat (Triticum aestivum L.) Seeds Proteome in Response to Anti–trx s Gene

    Guo, Hongxiang; Zhang, Huizhen; Li, Yongchun; Ren, Jiangping; Wang, Xiang; Niu, Hongbin; Yin, Jun

    2011-01-01

    Background Thioredoxin h (trx h) is closely related to germination of cereal seeds. The cDNA sequences of the thioredoxin s (trx s) gene from Phalaris coerulescens and the thioredoxin h (trx h) gene from wheat are highly homologous, and their expression products have similar biological functions. Transgenic wheat had been formed after the antisense trx s was transferred into wheat, and it had been certified that the expression of trx h decreased in transgenic wheat, and transgenic wheat has high resistance to pre-harvest sprouting. Methodology/Principal Findings Through analyzing the differential proteome of wheat seeds between transgenic wheat and wild type wheat, the mechanism of transgenic wheat seeds having high resistance to pre-harvest sprouting was studied in the present work. There were 36 differential proteins which had been identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All these differential proteins are involved in regulation of carbohydrates, esters, nucleic acid, proteins and energy metabolism, and biological stress. The quantitative real time PCR results of some differential proteins, such as trx h, heat shock protein 70, α-amylase, β-amylase, glucose-6-phosphate isomerase, 14-3-3 protein, S3-RNase, glyceraldehyde-3-phosphate dehydrogenase, and WRKY transcription factor 6, represented good correlation between transcripts and proteins. The biological functions of many differential proteins are consistent with the proposed role of trx h in wheat seeds. Conclusions/Significance A possible model for the role of trx h in wheat seeds germination was proposed in this paper. These results will not only play an important role in clarifying the mechanism that transgenic wheat has high resistance to pre-harvest sprouting, but also provide further evidence for the role of trx h in germination of wheat seeds. PMID:21811579

  10. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

  11. Identification of changes in wheat (Triticum aestivum L.) seeds proteome in response to anti-trx s gene.

    Guo, Hongxiang; Zhang, Huizhen; Li, Yongchun; Ren, Jiangping; Wang, Xiang; Niu, Hongbin; Yin, Jun

    2011-01-01

    Thioredoxin h (trx h) is closely related to germination of cereal seeds. The cDNA sequences of the thioredoxin s (trx s) gene from Phalaris coerulescens and the thioredoxin h (trx h) gene from wheat are highly homologous, and their expression products have similar biological functions. Transgenic wheat had been formed after the antisense trx s was transferred into wheat, and it had been certified that the expression of trx h decreased in transgenic wheat, and transgenic wheat has high resistance to pre-harvest sprouting. Through analyzing the differential proteome of wheat seeds between transgenic wheat and wild type wheat, the mechanism of transgenic wheat seeds having high resistance to pre-harvest sprouting was studied in the present work. There were 36 differential proteins which had been identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All these differential proteins are involved in regulation of carbohydrates, esters, nucleic acid, proteins and energy metabolism, and biological stress. The quantitative real time PCR results of some differential proteins, such as trx h, heat shock protein 70, α-amylase, β-amylase, glucose-6-phosphate isomerase, 14-3-3 protein, S3-RNase, glyceraldehyde-3-phosphate dehydrogenase, and WRKY transcription factor 6, represented good correlation between transcripts and proteins. The biological functions of many differential proteins are consistent with the proposed role of trx h in wheat seeds. A possible model for the role of trx h in wheat seeds germination was proposed in this paper. These results will not only play an important role in clarifying the mechanism that transgenic wheat has high resistance to pre-harvest sprouting, but also provide further evidence for the role of trx h in germination of wheat seeds.

  12. Proteomic Identification of Target Proteins of Thiodigalactoside in White Adipose Tissue from Diet-Induced Obese Rats

    Hilal Ahmad Parray

    2015-06-01

    Full Text Available Previously, galectin-1 (GAL1 was found to be up-regulated in obesity-prone subjects, suggesting that use of a GAL1 inhibitor could be a novel therapeutic approach for treatment of obesity. We evaluated thiodigalactoside (TDG as a potent inhibitor of GAL1 and identified target proteins of TDG by performing comparative proteome analysis of white adipose tissue (WAT from control and TDG-treated rats fed a high fat diet (HFD using two dimensional gel electrophoresis (2-DE combined with MALDI-TOF-MS. Thirty-two spots from a total of 356 matched spots showed differential expression between control and TDG-treated rats, as identified by peptide mass fingerprinting. These proteins were categorized into groups such as carbohydrate metabolism, tricarboxylic acid (TCA cycle, signal transduction, cytoskeletal, and mitochondrial proteins based on functional analysis using Protein Annotation Through Evolutionary Relationship (PANTHER and Database for Annotation, Visualization, Integrated Discovery (DAVID classification. One of the most striking findings of this study was significant changes in Carbonic anhydrase 3 (CA3, Voltage-dependent anion channel 1 (VDAC1, phosphatidylethanolamine-binding protein 1 (PEBP1, annexin A2 (ANXA2 and lactate dehydrogenase A chain (LDHA protein levels between WAT from control and TDG-treated groups. In addition, we confirmed increased expression of thermogenic proteins as well as reduced expression of lipogenic proteins in response to TDG treatment. These results suggest that TDG may effectively prevent obesity, and TDG-responsive proteins can be used as novel target proteins for obesity treatment.

  13. Identification of changes in wheat (Triticum aestivum L. seeds proteome in response to anti-trx s gene.

    Hongxiang Guo

    Full Text Available BACKGROUND: Thioredoxin h (trx h is closely related to germination of cereal seeds. The cDNA sequences of the thioredoxin s (trx s gene from Phalaris coerulescens and the thioredoxin h (trx h gene from wheat are highly homologous, and their expression products have similar biological functions. Transgenic wheat had been formed after the antisense trx s was transferred into wheat, and it had been certified that the expression of trx h decreased in transgenic wheat, and transgenic wheat has high resistance to pre-harvest sprouting. METHODOLOGY/PRINCIPAL FINDINGS: Through analyzing the differential proteome of wheat seeds between transgenic wheat and wild type wheat, the mechanism of transgenic wheat seeds having high resistance to pre-harvest sprouting was studied in the present work. There were 36 differential proteins which had been identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS. All these differential proteins are involved in regulation of carbohydrates, esters, nucleic acid, proteins and energy metabolism, and biological stress. The quantitative real time PCR results of some differential proteins, such as trx h, heat shock protein 70, α-amylase, β-amylase, glucose-6-phosphate isomerase, 14-3-3 protein, S3-RNase, glyceraldehyde-3-phosphate dehydrogenase, and WRKY transcription factor 6, represented good correlation between transcripts and proteins. The biological functions of many differential proteins are consistent with the proposed role of trx h in wheat seeds. CONCLUSIONS/SIGNIFICANCE: A possible model for the role of trx h in wheat seeds germination was proposed in this paper. These results will not only play an important role in clarifying the mechanism that transgenic wheat has high resistance to pre-harvest sprouting, but also provide further evidence for the role of trx h in germination of wheat seeds.

  14. Identification of candidate biomarkers of the exposure to PCBs in contaminated cattle: A gene expression- and proteomic-based approach.

    Girolami, F; Badino, P; Spalenza, V; Manzini, L; Renzone, G; Salzano, A M; Dal Piaz, F; Scaloni, A; Rychen, G; Nebbia, C

    2018-05-28

    Dioxins and polychlorinated biphenyls (PCBs) are widespread and persistent contaminants. Through a combined gene expression/proteomic-based approach, candidate biomarkers of the exposure to such environmental pollutants in cattle subjected to a real eco-contamination event were identified. Animals were removed from the polluted area and fed a standard ration for 6 months. The decontamination was monitored by evaluating dioxin and PCB levels in pericaudal fat two weeks after the removal from the contaminated area (day 0) and then bimonthly for six months (days 59, 125 and 188). Gene expression measurements demonstrated that CYP1B1 expression was significantly higher in blood lymphocytes collected in contaminated animals (day 0), and decreased over time during decontamination. mRNA levels of interleukin 2 showed an opposite quantitative trend. MALDI-TOF-MS polypeptide profiling of serum samples ascertained a progressive decrease (from day 0 to 188) of serum levels of fibrinogen β-chain and serpin A3-7-like fragments, apolipoprotein (APO) C-II and serum amyloid A-4 protein, along with an augmented representation of transthyretin isoforms, as well as APOC-III and APOA-II proteins during decontamination. When differentially represented species were combined with serum antioxidant, acute phase and proinflammatory protein levels already ascertained in the same animals (Cigliano et al., 2016), bioinformatics unveiled an interaction network linking together almost all components. This suggests the occurrence of a complex PCB-responsive mechanism associated with animal contamination/decontamination, including a cohort of protein/polypeptide species involved in blood redox homeostasis, inflammation and lipid transport. All together, these results suggest the use in combination of such biomarkers for identifying PCB-contaminated animals, and for monitoring the restoring of their healthy condition following a decontamination process. Copyright © 2018 Elsevier B.V. All

  15. Improved Understanding of Microbial Iron and Sulfate Reduction Through a Combination of Bottom-up and Top-down Functional Proteomics Assays

    Richardson, Ruth [Cornell Univ., Ithaca, NY (United States)

    2016-02-28

    Our overall goal was to improve the understanding of microbial iron and sulfate reduction by evaluating a diverse iron and sulfate reducing organisms utilizing a multi-omics approach combining “top-down” and “bottom-up” omics methodologies. We initiated one of the first combined comparative genomics, shotgun proteomics, RTqPCR, and heterologous expression studies in pursuit of our project objectives. Within the first year of this project, we created a new bioinformatics tool for ortholog identification (“SPOCS”). SPOCS is described in our publication, Curtis et al., 2013. Using this tool we were able to identify conserved orthologous groups across diverse iron and sulfate reducing microorganisms from Firmicutes, gamma-proteobacteria and delta-proteobacteria. For six iron and sulfate reducers we also performed shotgun proteomics (“bottom-up” proteomics including accurate mass and time (AMT) tag and iTRAQ approaches). Cultures include Gram (-) and Gram (+) microbes. Gram (-) were: Geobacter sulfureducens (grown on iron citrate and fumarate), Geobacter bemidjiensis (grown on iron citrate and fumarate), Shewanella oneidiensis (grown on iron citrate and fumarate) and Anaeromyxobacter dehalogenans (grown on iron citrate and fumarate). Although all cultures grew on insoluble iron, the iron precipitates interfered with protein extraction and analysis; which remains a major challenge for researchers in disparate study systems. Among the Gram (-) organisms studied, Anaeromyxobacter dehalogenans remains the most poorly characterized. Yet, it is arguably the most versatile organisms we studied. In this work we have used comparative proteomics to hypothesize which two of the dozens of predicted c-type cytochromes within Anaeromyxobacter dehalogenans may be directly involved in soluble iron reduction. Unfortunately, heterologous expression of these Anaeromyxobacter dehalogenans ctype cytochromes led to poor protein production and/or formation of inclusion bodies

  16. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  17. Arabidopsis peroxisome proteomics

    John D. Bussell

    2013-04-01

    Full Text Available The analytical depth of investigation of the peroxisomal proteome of the model plant Arabidopsis thaliana has not yet reached that of other major cellular organelles such as chloroplasts or mitochondria. This is primarily due to the difficulties associated with isolating and obtaining purified samples of peroxisomes from Arabidopsis. So far only a handful of research groups have been successful in obtaining such fractions. To make things worse, enriched peroxisome fractions frequently suffer from significant organellar contamination, lowering confidence in localization assignment of the identified proteins. As with other cellular compartments, identification of peroxisomal proteins forms the basis for investigations of the dynamics of the peroxisomal proteome. It is therefore not surprising that, in terms of functional analyses by proteomic means, there remains a considerable gap between peroxisomes and chloroplasts or mitochondria. Alternative strategies are needed to overcome the obstacle of hard-to-obtain organellar fractions. This will help to close the knowledge gap between peroxisomes and other organelles and provide a full picture of the physiological pathways shared between organelles. In this review we briefly summarize the status quo and discuss some of the methodological alternatives to classic organelle proteomic approaches.

  18. Xylem sap proteomics.

    de Bernonville, Thomas Dugé; Albenne, Cécile; Arlat, Matthieu; Hoffmann, Laurent; Lauber, Emmanuelle; Jamet, Elisabeth

    2014-01-01

    Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

  19. Proteomics dataset

    Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2017-01-01

    patients (Morgan et al., 2012; Abraham and Medzhitov, 2011; Bennike, 2014) [8–10. Therefore, we characterized the proteome of colon mucosa biopsies from 10 inflammatory bowel disease ulcerative colitis (UC) patients, 11 gastrointestinal healthy rheumatoid arthritis (RA) patients, and 10 controls. We...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

  20. Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration.

    Yuan Wang

    Full Text Available With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635.

  1. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-01-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

  2. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

    Zhen, Zhen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Luthringer, Bérengère, E-mail: berengere.luthringer@hzg.de [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Yang, Li [College of Engineering, Peking University, Beijing 100871 (China); Xi, Tingfei, E-mail: xitingfei@pku.edu.cn [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Zheng, Yufeng [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Feyerabend, Frank; Willumeit, Regine [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Lai, Chen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Ge, Zigang [College of Engineering, Peking University, Beijing 100871 (China)

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

  3. Proteome analysis of barley seeds: Identification of major proteins from two-dimensional gels (pl 4-7)

    Østergaard, O.; Finnie, Christine; Laugesen, S.

    2004-01-01

    inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin......Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water...

  4. Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

    Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

    2013-06-01

    The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.

  6. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highlydifferentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram

  7. Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

    Ghodasara, P; Sadowski, P; Satake, N; Kopp, S; Mills, P C

    2017-12-01

    Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

    Malenfant, Simon; Potus, François; Fournier, Frédéric; Breuils-Bonnet, Sandra; Pflieger, Aude; Bourassa, Sylvie; Tremblay, Ève; Nehmé, Benjamin; Droit, Arnaud; Bonnet, Sébastien; Provencher, Steeve

    2015-05-01

    Exercise limitation comes from a close interaction between cardiovascular and skeletal muscle impairments. To better understand the implication of possible peripheral oxidative metabolism dysfunction, we studied the proteomic signature of skeletal muscle in pulmonary arterial hypertension (PAH). Eight idiopathic PAH patients and eight matched healthy sedentary subjects were evaluated for exercise capacity, skeletal muscle proteomic profile, metabolism, and mitochondrial function. Skeletal muscle proteins were extracted, and fractioned peptides were tagged using an iTRAQ protocol. Proteomic analyses have documented a total of 9 downregulated proteins in PAH skeletal muscles and 10 upregulated proteins compared to healthy subjects. Most of the downregulated proteins were related to mitochondrial structure and function. Focusing on skeletal muscle metabolism and mitochondrial health, PAH patients presented a decreased expression of oxidative enzymes (pyruvate dehydrogenase, p metabolism in PAH skeletal muscles. We provide evidences that impaired mitochondrial and metabolic functions found in the lungs and the right ventricle are also present in skeletal muscles of patients. • Proteomic and metabolic analysis show abnormal oxidative metabolism in PAH skeletal muscle. • EM of PAH patients reveals abnormal mitochondrial structure and distribution. • Abnormal mitochondrial health and function contribute to exercise impairments of PAH. • PAH may be considered a vascular affliction of heart and lungs with major impact on peripheral muscles.

  9. Proteome-based bacterial identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical diagnostic microbiology.

    Nomura, Fumio

    2015-06-01

    Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for identification of microorganisms, including bacteria, yeast, molds, and mycobacteria. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. In the present review, the state of the art and advantages of MALDI-TOF MS-based bacterial identification are described. The potential of this innovative technology for use in strain typing and detection of antibiotic resistance is also discussed. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    and technologically challenging, and no ideal method is currently available. Here, we describe a strategy that allows scanning of the entire cell surface and identification of molecules that exhibit altered expression between two cell types. Concurrently, this method gives rise to valuable reagents for further...... characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic...... capabilities. Our results yielded a large panel of monoclonal antibodies (mAbs) that recognized cell surface markers preferentially or exclusively expressed on metastatic vs nonmetastatic cancer cells. Four mAbs and their corresponding antigens were further characterized. Importantly, analysis on an extended...

  11. Proteomic strategy for the identification of critical actors in reorganization of the post-meiotic male genome.

    Govin, Jerome; Gaucher, Jonathan; Ferro, Myriam; Debernardi, Alexandra; Garin, Jerome; Khochbin, Saadi; Rousseaux, Sophie

    2012-01-01

    After meiosis, during the final stages of spermatogenesis, the haploid male genome undergoes major structural changes, resulting in a shift from a nucleosome-based genome organization to the sperm-specific, highly compacted nucleoprotamine structure. Recent data support the idea that region-specific programming of the haploid male genome is of high importance for the post-fertilization events and for successful embryo development. Although these events constitute a unique and essential step in reproduction, the mechanisms by which they occur have remained completely obscure and the factors involved have mostly remained uncharacterized. Here, we sought a strategy to significantly increase our understanding of proteins controlling the haploid male genome reprogramming, based on the identification of proteins in two specific pools: those with the potential to bind nucleic acids (basic proteins) and proteins capable of binding basic proteins (acidic proteins). For the identification of acidic proteins, we developed an approach involving a transition-protein (TP)-based chromatography, which has the advantage of retaining not only acidic proteins due to the charge interactions, but also potential TP-interacting factors. A second strategy, based on an in-depth bioinformatic analysis of the identified proteins, was then applied to pinpoint within the lists obtained, male germ cells expressed factors relevant to the post-meiotic genome organization. This approach reveals a functional network of DNA-packaging proteins and their putative chaperones and sheds a new light on the way the critical transitions in genome organizations could take place. This work also points to a new area of research in male infertility and sperm quality assessments.

  12. Identification of Analytical Factors Affecting Complex Proteomics Profiles Acquired in a Factorial Design Study with Analysis of Variance : Simultaneous Component Analysis

    Mitra, V.; Govorukhina, N.; Zwanenburg, G.; Hoefsloot, H.; Westra, I.; Smilde, A.; Reijmers, T.; van der Zee, A.G.J.; Suits, F.; Bischoff, R.; Horvatovich, P.

    2016-01-01

    Complex shotgun proteomics peptide profiles obtained in quantitative differential protein expression studies, such as in biomarker discovery, may be affected by multiple experimental factors. These preanalytical factors may affect the measured protein abundances which in turn influence the outcome

  13. PIQMIe: A web server for semi-quantitative proteomics data management and analysis

    A. Kuzniar (Arnold); R. Kanaar (Roland)

    2014-01-01

    textabstractWe present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates

  14. Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

    Panjamaporn Sangwung

    Full Text Available Endothelial nitric oxide synthase (eNOS is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3 was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.

  15. High-resolution proteomic profiling of spider venom: expanding the toxin diversity of Phoneutria nigriventer venom.

    Liberato, Tarcísio; Troncone, Lanfranco Ranieri Paolo; Yamashiro, Edson T; Serrano, Solange M T; Zelanis, André

    2016-03-01

    Here we present a proteomic characterization of Phoneutria nigriventer venom. A shotgun proteomic approach allowed the identification, for the first time, of O-glycosyl hydrolases (chitinases) in P. nigriventer venom. The electrophoretic profiles under nonreducing and reducing conditions, and protein identification by mass spectrometry, indicated the presence of oligomeric toxin structures in the venom. Complementary proteomic approaches allowed for a qualitative and semi-quantitative profiling of P. nigriventer venom complexity, expanding its known venom proteome diversity.

  16. Quantitative proteomic analysis of host--pathogen interactions: a study of Acinetobacter baumannii responses to host airways.

    Méndez, Jose Antonio; Mateos, Jesús; Beceiro, Alejandro; Lopez, María; Tomás, María; Poza, Margarita; Bou, Germán

    2015-05-30

    Acinetobacter baumannii is a major health problem. The most common infection caused by A. baumannii is hospital acquired pneumonia, and the associated mortality rate is approximately 50%. Neither in vivo nor ex vivo expression profiling has been performed at the proteomic or transcriptomic level for pneumonia caused by A. baumannii. In this study, we characterized the proteome of A. baumannii under conditions that simulate those found in the airways, to gain some insight into how A. baumannii adapts to the host and to improve knowledge about the pathogenesis and virulence of this bacterium. A clinical strain of A. baumannii was grown under different conditions: in the presence of bronchoalveolar lavage fluid from infected rats, of RAW 264.7 cells to simulate conditions in the respiratory tract and in control conditions. We used iTRAQ labelling and LC-MALDI-TOF/TOF to investigate how A. baumannii responds on exposure to macrophages/BALF. 179 proteins showed differential expression. In both models, proteins involved in the following processes were over-expressed: (i) pathogenesis and virulence (OmpA, YjjK); (ii) cell wall/membrane/envelope biogenesis (MurC); (iii) energy production and conversion (acetyl-CoA hydrolase); and (iv) translation (50S ribosomal protein L9). Proteins involved in the following were under-expressed: (i) lipid metabolism (short-chain dehydrogenase); (ii) amino acid metabolism and transport (aspartate aminotransferase); (iii) unknown function (DNA-binding protein); and (iv) inorganic ion transport and metabolism (hydroperoxidase). We observed alterations in cell wall synthesis and identified 2 upregulated virulence-associated proteins with >15 peptides/protein in both ex vivo models (OmpA and YjjK), suggesting that these proteins are fundamental for pathogenesis and virulence in the airways. This study is the first comprehensive overview of the ex vivo proteome of A. baumannii and is an important step towards identification of diagnostic

  17. Scientific Workflow Management in Proteomics

    de Bruin, Jeroen S.; Deelder, André M.; Palmblad, Magnus

    2012-01-01

    Data processing in proteomics can be a challenging endeavor, requiring extensive knowledge of many different software packages, all with different algorithms, data format requirements, and user interfaces. In this article we describe the integration of a number of existing programs and tools in Taverna Workbench, a scientific workflow manager currently being developed in the bioinformatics community. We demonstrate how a workflow manager provides a single, visually clear and intuitive interface to complex data analysis tasks in proteomics, from raw mass spectrometry data to protein identifications and beyond. PMID:22411703

  18. Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

    Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.

    2009-01-01

    The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504

  19. Redox proteomics changes in the fungal pathogen Trichosporon asahii on arsenic exposure: identification of protein responses to metal-induced oxidative stress in an environmentally-sampled isolate.

    Sidra Ilyas

    Full Text Available Trichosporon asahii is a yeast pathogen implicated in opportunistic infections. Cultures of an isolate collected from industrial wastewater were exposed for 2 days to 100 mg/L sodium arsenite (NaAsO2 and cadmium (CdCl2. Both metals reduced glutathione transferase (GST activity but had no effect on superoxide dismutase or catalase. NaAsO2 exposure increased glutathione reductase activity while CdCl2 had no effect. Protein thiols were labeled with 5-iodoacetamido fluorescein followed by one dimensional electrophoresis which revealed extensive protein thiol oxidation in response to CdCl2 treatment but thiol reduction in response to NaAsO2. Two dimensional electrophoresis analyses showed that the intensity of some protein spots was enhanced on treatment as judged by SameSpots image analysis software. In addition, some spots showed decreased IAF fluorescence suggesting thiol oxidation. Selected spots were excised and tryptic digested for identification by MALDI-TOF/TOF MS. Twenty unique T. asahii proteins were identified of which the following proteins were up-regulated in response to NaAsO2: 3-isopropylmalate dehydrogenase, phospholipase B, alanine-glyoxylate aminotransferase, ATP synthase alpha chain, 20S proteasome beta-type subunit Pre3p and the hypothetical proteins A1Q1_08001, A1Q2_03020, A1Q1_06950, A1Q1_06913. In addition, the following showed decreased thiol-associated fluorescence consistent with thiol oxidation; aconitase; aldehyde reductase I; phosphoglycerate kinase; translation elongation factor 2; heat shock protein 70 and hypothetical protein A1Q2_04745. Some proteins showed both increase in abundance coupled with decrease in IAF fluorescence; 3-hydroxyisobutyryl-CoA hydrolase; homoserine dehydrogenase Hom6 and hypothetical proteins A1Q2_03020 and A1Q1_00754. Targets implicated in redox response included 10 unique metabolic enzymes, heat shock proteins, a component of the 20S proteasome and translation elongation factor 2. These data

  20. Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble γ-glutamyl transpeptidase

    Sajid Mohammed

    2006-05-01

    Full Text Available Background In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. Results Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a γ-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. γ-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG γ-glutamyl transpeptidase (GGT-1 is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong γ-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. Conclusion We have applied biochemical approaches, not used

  1. Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase.

    Walker, Michael J; Rylett, Caroline M; Keen, Jeff N; Audsley, Neil; Sajid, Mohammed; Shirras, Alan D; Isaac, R Elwyn

    2006-05-02

    In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a gamma-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. gamma-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG gamma-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong gamma-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. We have applied biochemical approaches, not used previously, to characterise

  2. Proteomics-grade de novo sequencing approach

    Savitski, Mikhail M; Nielsen, Michael L; Kjeldsen, Frank

    2005-01-01

    The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even kn...

  3. SuperQuant-assisted comparative proteome analysis of glioblastoma subpopulations allows for identification of potential novel therapeutic targets and cell markers

    Verano-Braga, Thiago; Gorshkov, Vladimir; Munthe, Sune

    2018-01-01

    Glioblastoma (GBM) is a highly aggressive brain cancer with poor prognosis and low survival rate. Invasive cancer stem-like cells (CSCs) are responsible for tumor recurrence because they escape current treatments. Our main goal was to study the proteome of three GBM subpopulations to identify key...... molecules behind GBM cell phenotypes and potential cell markers for migrating cells. We used SuperQuant-an enhanced quantitative proteome approach-to increase proteome coverage. We found 148 proteins differentially regulated in migrating CSCs and 199 proteins differentially regulated in differentiated cells...... migration. Moreover, our data suggested that microRNA-122 (miR-122) is a potential upstream regulator of GBM phenotypes as miR-122 activation was predicted for differentiated cells while its inhibition was predicted for migrating CSCs. Finally, we validated transferrin (TF) and procollagen-lysine 2...

  4. Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

    Coumans, Joëlle V F; Gau, David; Poljak, Anne; Wasinger, Valerie; Roy, Partha; Moens, Pierre D J

    2014-12-01

    Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer.

  5. Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

    Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

    2008-06-01

    Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

  6. Temporal differential proteomes of Clostridium difficile in the pig ileal-ligated loop model.

    Tavan Janvilisri

    Full Text Available The impact of Clostridium difficile infection (CDI on healthcare is becoming increasingly recognized as it represents a major cause of nosocomial diarrhea. A rising number of CDI cases and outbreaks have been reported worldwide. Here, we developed the pig ileal-ligated loop model for semi-quantitative analysis comparing temporal differential proteomes in C. difficile following in vivo incubation with in vitro growth using isobaric tags for relative and absolute quantification (iTRAQ. Proteins retrieved from the in vitro cultures and the loop contents after 4, 8, and 12 h in vivo incubation were subjected to in-solution digestion, iTRAQ labeling, two-dimensional liquid chromatography/tandem mass spectrometry and statistical analyses. From a total of 1152 distinct proteins identified in this study, 705 proteins were available for quantitative measures at all time points in both biological and technical replicates; 109 proteins were found to be differentially expressed. With analysis of clusters of orthologous group and protein-protein network interactions, we identified the proteins that might play roles in adaptive responses to the host environment, hence enhancing pathogenicity during CDI. This report represents the quantitative proteomic analysis of C. difficile that demonstrates time-dependent protein expression changes under conditions that mimic in vivo infection and identifies potential candidates for diagnostic or therapeutic measures.

  7. Quantitative proteomic analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hyphae differentiation

    Manteca, Angel; Sanchez, Jesus; Jung, Hye Ryung

    2010-01-01

    mycelial stages: an early compartmentalized vegetative mycelium (first mycelium, MI), and a multinucleated reproductive mycelium (second mycelium, MII), arising after PCD processes. In the present study, we made a detailed proteomic analysis of the distinct developmental stages of solid confluent...... Streptomyces coelicolor cultures using iTRAQ labelling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins...

  8. [Screening differentially expressed plasma proteins in cold stress rats based on iTRAQ combined with mass spectrometry technology].

    Liu, Yan-zhi; Guo, Jing-ru; Peng, Meng-ling; Ma, Li; Zhen, Li; Ji, Hong; Yang, Huan-min

    2015-09-01

    Isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry were used to screen differentially expressed plasma proteins in cold stress rats. Thirty health SPF Wistar rats were randomly divided into cold stress group A and control group B, then A and B were randomly divided into 3 groups (n = 5): A1, A2, A3 and B1, B2, B3. The temperature of room raising was (24.0 +/- 0.1) degrees C, and the cold stress temperature was (4.0 +/- 0.1) degrees C. The rats were treated with different temperatures until 12 h. The abdominal aortic blood was collected with heparin anticoagulation suction tube. Then, the plasma was separated for protein extraction, quantitative, enzymolysis, iTHAQ labeling, scx fractionation and mass spectrometry analysis. Totally, 1085 proteins were identified in the test, 39 differentially expressed proteins were screened, including 29 up-regulated proteins and 10 down-regulated proteins. Three important differentially expressed proteins related to cold stress were screened by bioinfonnatics analysis (Minor histocompatihility protein HA-1, Has-related protein Rap-1b, Integrin beta-1). In the experiment, the differentially expressed plasma proteins were successfully screened in cold stress rats. iTRAQ technology provided a good platform to screen protein diaguostic markers on cold stress rats, and laid a good foundation for further. study on animal cold stress mechanism.

  9. Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

    Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2011-01-01

    To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A high-quality catalog of the Drosophila melanogaster proteome

    Brunner, Erich; Ahrens, Christian H.; Mohanty, Sonaly

    2007-01-01

    % of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis...

  11. Aspects of the barley seed proteome during development and germination

    Finnie, Christine; Maeda, K.; Østergaard, O.

    2004-01-01

    Analysis of the water-soluble barley seed proteome has led to the identification of proteins by MS in the major spots on two-dimensional gels covering the pi ranges 4-7 and 6-11. This provides the basis for in-depth studies of proteome changes during seed development and germination, tissue...

  12. The Seed Proteome Web Portal

    Marc eGalland

    2012-06-01

    Full Text Available The Seed Proteome Web Portal (SPWP; http://www.seedproteome.com/ gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from 2 dimensional electrophoresis (2DE maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35% or a decreasing abundance (15%. Moreover, during radicle protrusion (24 h to 48 h upon imbibition, 41% proteins display quantitative variations with an increased (23% or a decreasing abundance (18%. In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r2 = 0.29 between the theoretical (predicted from Arabidopsis genome and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthetized proteins radiolabeled by [35S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

  13. Proteomic analysis of embryonic axis of Pisum sativum seeds during germination and identification of proteins associated with loss of desiccation tolerance

    Wang, Wei-Qing; Møller, Ian Max; Song, Song-Quan

    2012-01-01

    Seed germination is an important stage in life cycle of higher plants. The germination processes and its associated loss of desiccation tolerance, however, are still poorly understood. In present study, pea seeds were used to study changes in embryonic axis proteome during germination by 2-DE...... and mass spectrometry. We identified a total of 139 protein spots showing a significant (>2-fold) change during germination. The results show that seed germination is not only the activation of a series of metabolic processes, but also involves reorganization of cellular structure and activation...... of protective systems. To uncouple the physiological processes of germination and its associated loss of desiccation tolerance, we used the fact that pea seeds have different desiccation tolerance when imbibed in water, CaCl2 and methylviologen at the same germination stage. We compared the proteome amongst...

  14. Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein

    Vrablik, Tracy L. [Washington State Univ., Pullman, WA (United States); Petyuk, Vladislav A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Larson, Emily M. [Washington State Univ., Pullman, WA (United States); Smith, Richard D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Watts, Jennifer [Washington State Univ., Pullman, WA (United States)

    2015-06-27

    Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified C. elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols was rich in fatty acid species obtained from the dietary E. coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a relative decrease of MDT-28 abundance in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans.

  15. Shot-gun proteome and transcriptome mapping of the jujube floral organ and identification of a pollen-specific S-locus F-box gene

    Ruihong Chen

    2017-07-01

    Full Text Available The flower is a plant reproductive organ that forms part of the fruit produced as the flowering season ends. While the number and identity of proteins expressed in a jujube (Ziziphus jujuba Mill. flower is currently unknown, integrative proteomic and transcriptomic analyses provide a systematic strategy of characterizing the floral biology of plants. We conducted a shotgun proteomic analysis on jujube flowers by using a filter-aided sample preparation tryptic digestion, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS. In addition, transcriptomics analyses were performed on HiSeq2000 sequencers. In total, 7,853 proteins were identified accounting for nearly 30% of the ‘Junzao’ gene models (27,443. Genes identified in proteome generally showed higher RPKM (reads per kilobase per million mapped reads values than undetected genes. Gene ontology categories showed that ribosomes and intracellular organelles were the most dominant classes and accounted for 17.0% and 14.0% of the proteome mass, respectively. The top-ranking proteins with iBAQ >1010 included non-specific lipid transfer proteins, histones, actin-related proteins, fructose-bisphosphate aldolase, Bet v I type allergens, etc. In addition, we identified one pollen-specificity S-locus F-box-like gene located on the same chromosome as the S-RNase gene. Both of these may activate the behaviour of gametophyte self-incompatibility in jujube. These results reflected the protein profile features of jujube flowers and contributes new information important to the jujube breeding system.

  16. Comparison of theoretical proteomes: identification of COGs with conserved and variable pI within the multimodal pI distribution.

    Nandi, Soumyadeep; Mehra, Nipun; Lynn, Andrew M; Bhattacharya, Alok

    2005-09-09

    Theoretical proteome analysis, generated by plotting theoretical isoelectric points (pI) against molecular masses of all proteins encoded by the genome show a multimodal distribution for pI. This multimodal distribution is an effect of allowed combinations of the charged amino acids, and not due to evolutionary causes. The variation in this distribution can be correlated to the organisms ecological niche. Contributions to this variation maybe mapped to individual proteins by studying the variation in pI of orthologs across microorganism genomes. The distribution of ortholog pI values showed trimodal distributions for all prokaryotic genomes analyzed, similar to whole proteome plots. Pairwise analysis of pI variation show that a few COGs are conserved within, but most vary between, the acidic and basic regions of the distribution, while molecular mass is more highly conserved. At the level of functional grouping of orthologs, five groups vary significantly from the population of orthologs, which is attributed to either conservation at the level of sequences or a bias for either positively or negatively charged residues contributing to the function. Individual COGs conserved in both the acidic and basic regions of the trimodal distribution are identified, and orthologs that best represent the variation in levels of the acidic and basic regions are listed. The analysis of pI distribution by using orthologs provides a basis for resolution of theoretical proteome comparison at the level of individual proteins. Orthologs identified that significantly vary between the major acidic and basic regions maybe used as representative of the variation of the entire proteome.

  17. Comparison of theoretical proteomes: Identification of COGs with conserved and variable pI within the multimodal pI distribution

    Lynn Andrew M

    2005-09-01

    Full Text Available Abstract Background Theoretical proteome analysis, generated by plotting theoretical isoelectric points (pI against molecular masses of all proteins encoded by the genome show a multimodal distribution for pI. This multimodal distribution is an effect of allowed combinations of the charged amino acids, and not due to evolutionary causes. The variation in this distribution can be correlated to the organisms ecological niche. Contributions to this variation maybe mapped to individual proteins by studying the variation in pI of orthologs across microorganism genomes. Results The distribution of ortholog pI values showed trimodal distributions for all prokaryotic genomes analyzed, similar to whole proteome plots. Pairwise analysis of pI variation show that a few COGs are conserved within, but most vary between, the acidic and basic regions of the distribution, while molecular mass is more highly conserved. At the level of functional grouping of orthologs, five groups vary significantly from the population of orthologs, which is attributed to either conservation at the level of sequences or a bias for either positively or negatively charged residues contributing to the function. Individual COGs conserved in both the acidic and basic regions of the trimodal distribution are identified, and orthologs that best represent the variation in levels of the acidic and basic regions are listed. Conclusion The analysis of pI distribution by using orthologs provides a basis for resolution of theoretical proteome comparison at the level of individual proteins. Orthologs identified that significantly vary between the major acidic and basic regions maybe used as representative of the variation of the entire proteome.

  18. Proteomic-based identification of multiple pathways underlying n-butylidenephthalide-induced apoptosis in LNCaP human prostate cancer cells.

    Pang, Cheng-Yoong; Chiu, Sheng-Chun; Harn, Horng-Jyh; Zhai, Wei-Jun; Lin, Shinn-Zong; Yang, Hsueh-Hui

    2013-09-01

    Although numerous studies have shown the cancer-preventive properties of butylidenephthalide (BP), there is little report of BP affecting human prostate cancer cells. In the present study, proteomic-based approaches were used to elucidate the anticancer mechanism of BP in LNCaP human prostate cancer cells. BP treatment decreased the viability of LNCaP human prostate cancer cells in a concentration- and time-dependent manner, which was correlated with G0/G1 phase cell cycle arrest. Increased cell cycle arrest was associated with a decrease in the level of CCND1, CDK2, and PCNA proteins and an increase in the level of CDKN2A, CDKN1A, and SFN proteins. Proteomic studies revealed that among 48 differentially expressed proteins, 25 proteins were down-regulated and 23 proteins were up-regulated and these proteins fall into one large protein protein interaction network. Among these proteins, FAS, AIFM1, BIK, CYCS, SFN, PPP2R1A, CALR, HSPA5, DDIT3, and ERN1 are apoptosis and endoplasmic reticulum (ER) stress associated proteins. Proteomic data suggested that multiple signaling pathways including FAS-dependent pathway, mitochondrial pathway, and ER stress pathway are involved in the apoptosis induced by BP. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  20. Proteomic Analysis of Lung Tissue in a Rat Acute Lung Injury Model: Identification of PRDX1 as a Promoter of Inflammation

    Dongdong Liu

    2014-01-01

    Full Text Available Acute respiratory distress syndrome (ARDS remains a high morbidity and mortality disease entity in critically ill patients, despite decades of numerous investigations into its pathogenesis. To obtain global protein expression changes in acute lung injury (ALI lung tissues, we employed a high-throughput proteomics method to identify key components which may be involved in the pathogenesis of ALI. In the present study, we analyzed lung tissue proteomes of Pseudomonas aeruginosa-induced ALI rats and identified eighteen proteins whose expression levels changed more than twofold as compared to normal controls. In particular, we found that PRDX1 expression in culture medium was elevated by a lipopolysaccharide (LPS challenge in airway epithelial cells in vitro. Furthermore, overexpression of PRDX1 increased the expression of proinflammatory cytokines interleukin-6 (IL-6, interleukin-8 (IL-8, and tumor necrosis factor-α (TNF-α, whereas knockdown of PRDX1 led to downregulated expression of cytokines induced by LPS. In conclusion, our findings provide a global alteration in the proteome of lung tissues in the ALI rat model and indicate that PRDX1 may play a critical role in the pathogenesis of ARDS by promoting inflammation and represent a novel strategy for the development of new therapies against ALI.

  1. Proteogenomics Dashboard for the Human Proteome Project.

    Tabas-Madrid, Daniel; Alves-Cruzeiro, Joao; Segura, Victor; Guruceaga, Elizabeth; Vialas, Vital; Prieto, Gorka; García, Carlos; Corrales, Fernando J; Albar, Juan Pablo; Pascual-Montano, Alberto

    2015-09-04

    dasHPPboard is a novel proteomics-based dashboard that collects and reports the experiments produced by the Spanish Human Proteome Project consortium (SpHPP) and aims to help HPP to map the entire human proteome. We have followed the strategy of analog genomics projects like the Encyclopedia of DNA Elements (ENCODE), which provides a vast amount of data on human cell lines experiments. The dashboard includes results of shotgun and selected reaction monitoring proteomics experiments, post-translational modifications information, as well as proteogenomics studies. We have also processed the transcriptomics data from the ENCODE and Human Body Map (HBM) projects for the identification of specific gene expression patterns in different cell lines and tissues, taking special interest in those genes having little proteomic evidence available (missing proteins). Peptide databases have been built using single nucleotide variants and novel junctions derived from RNA-Seq data that can be used in search engines for sample-specific protein identifications on the same cell lines or tissues. The dasHPPboard has been designed as a tool that can be used to share and visualize a combination of proteomic and transcriptomic data, providing at the same time easy access to resources for proteogenomics analyses. The dasHPPboard can be freely accessed at: http://sphppdashboard.cnb.csic.es.

  2. Polyphemus, Odysseus and the ovine milk proteome.

    Cunsolo, Vincenzo; Fasoli, Elisa; Di Francesco, Antonella; Saletti, Rosaria; Muccilli, Vera; Gallina, Serafina; Righetti, Pier Giorgio; Foti, Salvatore

    2017-01-30

    In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier . Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

    Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

    2015-04-21

    A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

  4. Proteomic and metabolomic approaches to biomarker discovery

    Issaq, Haleem J

    2013-01-01

    Proteomic and Metabolomic Approaches to Biomarker Discovery demonstrates how to leverage biomarkers to improve accuracy and reduce errors in research. Disease biomarker discovery is one of the most vibrant and important areas of research today, as the identification of reliable biomarkers has an enormous impact on disease diagnosis, selection of treatment regimens, and therapeutic monitoring. Various techniques are used in the biomarker discovery process, including techniques used in proteomics, the study of the proteins that make up an organism, and metabolomics, the study of chemical fingerprints created from cellular processes. Proteomic and Metabolomic Approaches to Biomarker Discovery is the only publication that covers techniques from both proteomics and metabolomics and includes all steps involved in biomarker discovery, from study design to study execution.  The book describes methods, and presents a standard operating procedure for sample selection, preparation, and storage, as well as data analysis...

  5. Characterization of individual mouse cerebrospinal fluid proteomes

    Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles; Orton, Daniel J.; Moore, Ronald J.; Smith, Richard D.

    2014-03-20

    Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% false discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.

  6. Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings

    Zhujia Ye

    2016-08-01

    Full Text Available Switchgrass (Panicum virgatum is a perennial crop producing deep roots and thus highly tolerant to soil water deficit conditions. However, seedling establishment in the field is very susceptible to prolonged and periodic drought stress. In this study, a “sandwich” system simulating a gradual water deletion process was developed. Switchgrass seedlings were subjected to a 20-day gradual drought treatment process when soil water tension was increased to 0.05 MPa (moderate drought stress and leaf physiological properties had expressed significant alteration. Drought-induced changes in leaf proteomes were identified using the isobaric tags for relative and absolute quantitation (iTRAQ labeling method followed by nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS analysis. Additionally, total leaf proteins were processed using a combinatorial library of peptide ligands to enrich for lower abundance proteins. Both total proteins and those enriched samples were analyzed to increase the coverage of the quantitative proteomics analysis. A total of 7006 leaf proteins were identified, and 257 (4% of the leaf proteome expressed a significant difference (p < 0.05, fold change <0.6 or >1.7 from the non-treated control to drought-treated conditions. These proteins are involved in the regulation of transcription and translation, cell division, cell wall modification, phyto-hormone metabolism and signaling transduction pathways, and metabolic pathways of carbohydrates, amino acids, and fatty acids. A scheme of abscisic acid (ABA-biosynthesis and ABA responsive signal transduction pathway was reconstructed using these drought-induced significant proteins, showing systemic regulation at protein level to deploy the respective mechanism. Results from this study, in addition to revealing molecular responses to drought stress, provide a large number of proteins (candidate genes that can be employed to improve switchgrass seedling growth and

  7. The Urine Proteome Profile Is Different in Neuromyelitis Optica Compared to Multiple Sclerosis: A Clinical Proteome Study.

    Helle H Nielsen

    Full Text Available Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO, NMO spectrum disorders (NMO-SD and multiple sclerosis (MS. Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS.The proteins in urine samples from anti-aquaporin 4 (AQP4 seropositive NMO/NMO-SD patients (n = 32, patients with MS (n = 46 and healthy subjects (HS, n = 31 were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig in the urine were validated by nephelometry in an independent cohort (n = 9-10 pr. groups.The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002. Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD.The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS.

  8. Urine Proteomics in the Era of Mass Spectrometry

    Ashley Beasley-Green

    2016-11-01

    Full Text Available With the technological advances of mass spectrometry (MS-based platforms, clinical proteomics is one of the most rapidly growing areas in biomedical research. Urine proteomics has become a popular subdiscipline of clinical proteomics because it is an ideal source for the discovery of noninvasive disease biomarkers. The urine proteome offers a comprehensive view of the local and systemic physiology since the proteome is primarily composed of proteins/peptides from the kidneys and plasma. The emergence of MS-based proteomic platforms as prominent bioanalytical tools in clinical applications has enhanced the identification of protein-based urinary biomarkers. This review highlights the characteristics of urine that make it an attractive biofluid for biomarker discovery and the impact of MS-based technologies on the clinical assessment of urinary protein biomarkers.

  9. Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal.

    Scholl, P F; Cole, R N; Ruczinski, I; Gucek, M; Diez, R; Rennie, A; Nathasingh, C; Schulze, K; Christian, P; Yager, J D; Groopman, J D; West, K P

    2012-05-01

    Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers. In 23 Nepalese pregnant women who delivered at term, two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2-6.8) exhibited nominally significant (p < 0.05) differences in abundance across trimesters. Additional filtering using a Bonferroni correction reduced the number of significant (p < 0.00019) spots to 61. Mass spectrometric analysis detected 38 proteins associated with gestational age, cytoskeletal remodeling, blood pressure regulation, lipid and nutrient transport, and inflammation. One new protein, pregnancy-specific β-glycoprotein 4 was detected. A follow-up isotope tagging for relative and absolute quantitation (iTRAQ) experiment of six mothers from the DIGE study revealed 111 proteins, of which 11 exhibited significant (p < 0.05) differences between trimesters. Four of these proteins: gelsolin, complement C1r subcomponent, α-1-acid glycoprotein, and α-1B-glycoprotein also changed in the DIGE analysis. Although not previously associated with normal pregnancy, gelsolin decreased in abundance by the third trimester (p < 0.01) in DIGE, iTRAQ and Western analyses. Changes in abundance of proteins in serum that are associated with syncytiotrophoblasts (gelsolin, pregnancy-specific β-1 glycoprotein 1 and β-2-glycoprotein I) probably reflect dynamics of a placental proteome shed into maternal circulation during pregnancy. Measurement of changes in the maternal serum proteome, when linked with birth outcomes, may yield biomarkers for tracking reproductive health in resource poor settings in future studies. Published by Elsevier Ltd.

  10. Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

    Wang, Zhen; Schey, Kevin L

    2015-12-01

    Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

  11. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

    2017-09-01

    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  12. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. Copyright © 2016. Published by Elsevier B.V.

  13. Comparative proteome analysis of human epithelial ovarian cancer

    Gagné Jean-Philippe

    2007-09-01

    Full Text Available Abstract Background Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer. Results The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior. Conclusion The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.

  14. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  15. A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

    Owens, RA; Hammel, S; Sheridan, KJ; Jones, GW; Doyle, S

    2014-01-01

    A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Ind...

  16. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Paul, Debasish; Kumar, Avinash; Gajbhiye, Akshada; Santra, Manas K.; Srikanth, Rapole

    2013-01-01

    Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches. PMID:23586059

  17. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Debasish Paul

    2013-01-01

    Full Text Available Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

  18. Mining the granule proteome

    Albrethsen, Jakob; Goetze, Jens P; Johnsen, Anders H

    2015-01-01

    Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of g...

  19. Analysis of Peanut Leaf Proteome

    Ramesh, R.; Suravajhala, Prashanth; Pechan, T.

    2010-01-01

    Peanut (Arachis hypogaea) is one of the most important sources of plant protein. Current selection of genotypes requires molecular characterization of available populations. Peanut genome database has several EST cDNAs which can be used to analyze gene expression. Analysis of proteins is a direct...... approach to define function of their associated genes. Proteome analysis linked to genome sequence information is critical for functional genomics. However, the available protein expression data is extremely inadequate. Proteome analysis of peanut leaf was conducted using two-dimensional gel...... electrophoresis in combination with sequence identification using MALDI/TOF to determine their identity and function related to growth, development and responses to stresses. Peanut leaf proteins were resolved into 300 polypeptides with pI values between 3.5 and 8.0 and relative molecular masses from 12 to 100 k...

  20. Birth of plant proteomics in India: a new horizon.

    Narula, Kanika; Pandey, Aarti; Gayali, Saurabh; Chakraborty, Niranjan; Chakraborty, Subhra

    2015-09-08

    In the post-genomic era, proteomics is acknowledged as the next frontier for biological research. Although India has a long and distinguished tradition in protein research, the initiation of proteomics studies was a new horizon. Protein research witnessed enormous progress in protein separation, high-resolution refinements, biochemical identification of the proteins, protein-protein interaction, and structure-function analysis. Plant proteomics research, in India, began its journey on investigation of the proteome profiling, complexity analysis, protein trafficking, and biochemical modeling. The research article by Bhushan et al. in 2006 marked the birth of the plant proteomics research in India. Since then plant proteomics studies expanded progressively and are now being carried out in various institutions spread across the country. The compilation presented here seeks to trace the history of development in the area during the past decade based on publications till date. In this review, we emphasize on outcomes of the field providing prospects on proteomic pathway analyses. Finally, we discuss the connotation of strategies and the potential that would provide the framework of plant proteome research. The past decades have seen rapidly growing number of sequenced plant genomes and associated genomic resources. To keep pace with this increasing body of data, India is in the provisional phase of proteomics research to develop a comparative hub for plant proteomes and protein families, but it requires a strong impetus from intellectuals, entrepreneurs, and government agencies. Here, we aim to provide an overview of past, present and future of Indian plant proteomics, which would serve as an evaluation platform for those seeking to incorporate proteomics into their research programs. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Quantitative proteomic analysis of the rice (Oryza sativa L. salt response.

    Jianwen Xu

    Full Text Available Salt stress is one of most serious limiting factors for crop growth and production. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ approach was used to analyze proteomic changes in rice shoots under salt stress in this study. A total of 56 proteins were significantly altered and 16 of them were enriched in the pathways of photosynthesis, antioxidant and oxidative phosphorylation. Among these 16 proteins, peroxiredoxin Q and photosystem I subunit D were up-regulated, while thioredoxin M-like, thioredoxin x, thioredoxin peroxidase, glutathione S-transferase F3, PSI subunit H, light-harvesting antenna complex I subunits, chloroplast chaperonin, vacuolar ATP synthase subunit H, and ATP synthase delta chain were down-regulated. Moreover, physiological data including total antioxidant capacity, peroxiredoxin activity, chlorophyll a/b content, glutathione S-transferase activity, reduced glutathione content and ATPase activity were consistent with changes in the levels of these proteins. The levels of the mRNAs encoding these proteins were also analyzed by real-time quantitative reverse transcription PCR, and approximately 86% of the results were consistent with the iTRAQ data. Importantly, our data suggest the important role of PSI in balancing energy supply and ROS generation under salt stress. This study provides information for an improved understanding of the function of photosynthesis and PSI in the salt-stress response of rice.

  2. A Proteomic Approach to Investigate the Drought Response in the Orphan Crop Eragrostis tef

    Rizqah Kamies

    2017-11-01

    Full Text Available The orphan crop, Eragrostis tef, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants tolerated water loss to 50% relative water content (RWC before adverse effects in leaf tissues were observed. Proteomic analysis using isobaric tag for relative and absolute quantification (iTRAQ mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with the imposed stress conditions. Validation of the iTRAQ dataset was done with selected stress-related proteins, fructose-bisphosphate aldolase (FBA and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR and peroxidase (POX. Western blot analyses confirmed protein presence and showed increased protein abundance levels during water deficit while enzymatic activity for FBA, MDHAR and POX increased at selected RWC points. Gene ontology (GO-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef upregulated proteins, while terms linked to reactive oxygen species (ROS-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef downregulated proteins.

  3. A Proteomic Approach to Investigate the Drought Response in the Orphan Crop Eragrostis tef.

    Kamies, Rizqah; Farrant, Jill M; Tadele, Zerihun; Cannarozzi, Gina; Rafudeen, Mohammed Suhail

    2017-11-15

    The orphan crop, Eragrostis tef , was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants tolerated water loss to 50% relative water content (RWC) before adverse effects in leaf tissues were observed. Proteomic analysis using isobaric tag for relative and absolute quantification (iTRAQ) mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with the imposed stress conditions. Validation of the iTRAQ dataset was done with selected stress-related proteins, fructose-bisphosphate aldolase (FBA) and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR) and peroxidase (POX). Western blot analyses confirmed protein presence and showed increased protein abundance levels during water deficit while enzymatic activity for FBA, MDHAR and POX increased at selected RWC points. Gene ontology (GO)-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef upregulated proteins, while terms linked to reactive oxygen species (ROS)-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef downregulated proteins.

  4. Minimally-invasive, microneedle-array extraction of interstitial fluid for comprehensive biomedical applications: transcriptomics, proteomics, metabolomics, exosome research, and biomarker identification.

    Taylor, Robert M; Miller, Philip R; Ebrahimi, Parwana; Polsky, Ronen; Baca, Justin T

    2018-01-01

    Interstitial fluid (ISF) has recently garnered interest as a biological fluid that could be used as an alternate to blood for biomedical applications, diagnosis, and therapy. ISF extraction techniques are promising because they are less invasive and less painful than venipuncture. ISF is an alternative, incompletely characterized source of physiological data. Here, we describe a novel method of ISF extraction in rats, using microneedle arrays, which provides volumes of ISF that are sufficient for downstream analysis techniques such as proteomics, genomics, and extracellular vesicle purification and analysis. This method is potentially less invasive than previously reported techniques. The limited invasiveness and larger volumes of extracted ISF afforded by this microneedle-assisted ISF extraction method provide a technique that is less stressful and more humane to laboratory animals, while also allowing for a reduction in the numbers of animals needed to acquire sufficient volumes of ISF for biomedical analysis and application.

  5. [Proteomics and transfusion medicine].

    Lion, N; Prudent, M; Crettaz, D; Tissot, J-D

    2011-04-01

    The term "proteomics" covers tools and techniques that are used to analyze and characterize complex mixtures of proteins from various biological samples. In this short review, a typical proteomic approach, related to the study of particular and illustrative situation related to transfusion medicine is reported. This "case report" will allow the reader to be familiar with a practical proteomic approach of a real situation, and will permit to describe the tools that are usually used in proteomic labs, and, in a second part, to present various proteomic applications in transfusion medicine. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  6. Virtual Labs in proteomics: new E-learning tools.

    Ray, Sandipan; Koshy, Nicole Rachel; Reddy, Panga Jaipal; Srivastava, Sanjeeva

    2012-05-17

    Web-based educational resources have gained enormous popularity recently and are increasingly becoming a part of modern educational systems. Virtual Labs are E-learning platforms where learners can gain the experience of practical experimentation without any direct physical involvement on real bench work. They use computerized simulations, models, videos, animations and other instructional technologies to create interactive content. Proteomics being one of the most rapidly growing fields of the biological sciences is now an important part of college and university curriculums. Consequently, many E-learning programs have started incorporating the theoretical and practical aspects of different proteomic techniques as an element of their course work in the form of Video Lectures and Virtual Labs. To this end, recently we have developed a Virtual Proteomics Lab at the Indian Institute of Technology Bombay, which demonstrates different proteomics techniques, including basic and advanced gel and MS-based protein separation and identification techniques, bioinformatics tools and molecular docking methods, and their applications in different biological samples. This Tutorial will discuss the prominent Virtual Labs featuring proteomics content, including the Virtual Proteomics Lab of IIT-Bombay, and E-resources available for proteomics study that are striving to make proteomic techniques and concepts available and accessible to the student and research community. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 14). Details can be found at: http://www.proteomicstutorials.org/. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome.

    Jason T Popesku

    2010-08-01

    Full Text Available Dopamine (DA is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA.Female goldfish were injected (i.p. with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555 and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively.The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system.

  8. Proteome analysis of Aspergillus ochraceus.

    Rizwan, Muhammad; Miller, Ingrid; Tasneem, Fareeha; Böhm, Josef; Gemeiner, Manfred; Razzazi-Fazeli, Ebrahim

    2010-08-01

    Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.

  9. Spermatogenesis in mammals: proteomic insights.

    Chocu, Sophie; Calvel, Pierre; Rolland, Antoine D; Pineau, Charles

    2012-08-01

    Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling

  10. Urine, peritoneal fluid and omental fat proteomes of reproductive age women: Endometriosis-related changes and associations with endocrine disrupting chemicals.

    Williams, Katherine E; Miroshnychenko, Olga; Johansen, Eric B; Niles, Richard K; Sundaram, Rajeshwari; Kannan, Kurunthachalam; Albertolle, Matthew; Zhou, Yan; Prasad, Namrata; Drake, Penelope M; Giudice, Linda C; Hall, Steven C; Witkowska, H Ewa; Buck Louis, Germaine M; Fisher, Susan J

    2015-01-15

    Endometriosis, ectopic growth of the uterine lining (endometrium), which affects 6-11% of reproductive age women, is associated with pelvic pain and infertility. We investigated the peritoneal fluid (PF), urine and omental fat (OF) proteomes of women with endometriosis vs. individuals with no surgically visualized endometriosis. All participants were enrolled in the NICHD-funded ENDO Study. A two-step proteomic study was performed. The first, a broad survey, employed a semi-quantitative gel LC-mass spectrometry (MS) workflow: SDS PAGE fractionation, trypsin digestion and LC-MS/MS. The results showed sample integrity but failed to detect any differences between women with and without endometriosis. The second step was a quantitative analysis of OF samples. We employed another sample set (n=30) from women ± disease and isobaric mass-tag (iTRAQ) chemistry to label peptides and 2D LC-MS/MS for protein identification and quantification. Three proteins-matrix metalloproteinase-9, neutrophil elastase, and FAM49B-were significantly lower in abundance in samples from women with endometriosis. Interestingly, neutrophil elastase and FAM49B levels were associated with higher levels of a subset of endocrine disrupting chemicals (EDCs) that were previously measured in the same samples. The results of these experiments showed the feasibility of associating endometriosis with changes in the OF protein repertoire and EDC levels. Endometriosis, pathological growth of the uterine lining, is associated with significant morbidities, including pain and infertility. However, the causes of this common condition are poorly understood. This study determined whether endometriosis was associated with changes in the protein composition of peritoneal fluid, urine and/or omental fat. A protein of unknown function (FAM49B) and two proteinases (metalloproteinase-9, neutrophil elastase) were down regulated in OF samples from women with versus without endometriosis. These findings suggested

  11. Proteomic Analysis of Plasma-Purified VLDL, LDL, and HDL Fractions from Atherosclerotic Patients Undergoing Carotid Endarterectomy: Identification of Serum Amyloid A as a Potential Marker

    Antonio J. Lepedda

    2013-01-01

    Full Text Available Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.

  12. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  13. Proteomic Identification of an Upregulated Isoform of Annexin A3 in the Spinal Cords of Rats in a Neuropathic Pain Model

    Wangyuan Zou

    2017-09-01

    Full Text Available Neuropathic pain (NP is induced by nerve damage or a disturbance in the peripheral or central nervous systems. Nerve damage causes the activation of sensitizing mechanisms in the peripheral and central nervous systems, which induces transcriptional and post-transcriptional alterations in sensory nerves. However, the underlying mechanisms of NP remain elusive. In the study, Two-dimensional gel electrophoresis (2DGE-based comparative proteomics identified 38 differential gel spots, and 15 differentially expressed proteins (DEPs between the sham and the chronic constriction injury (CCI-induced neuropathic pain rats. Of them, Annexin A3 (ANXA3 was significantly increased after CCI with Western blot analysis and immunofluorescence imaging. A lentivirus delivering ANXA3 shRNA (LV-shANXA3 was administered intrathecally to determine the analgesic effects of ANXA3 on allodynia and hyperalgesia in a CCI-induced neuropathic pain model in rats. Further study showed that LV-shANXA3 reversed the upregulation of ANXA3, alleviated CCI-induced mechanical allodynia and thermal hyperalgesia. The study indicated that ANXA3 may play an important role in neuropathic pain.

  14. Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp. bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677.

    Zhai, Zhengyuan; Douillard, François P; An, Haoran; Wang, Guohong; Guo, Xinghua; Luo, Yunbo; Hao, Yanling

    2014-06-01

    To overcome the deleterious effects of acid stress, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) elicits an adaptive response to acid stress. In this study, proteomics approach complemented by transcriptional analysis revealed some cellular changes in L. bulgaricus CAUH1 during acid adaptation. We observed an increase of glycolysis-associated proteins, promoting an optimal utilization of carbohydrates. Also, rerouting of the pyruvate metabolism to fatty acid biosynthesis was observed, indicating a possible modification of the cell membrane rigidity and impermeability. In addition, expression of ribosomal protein S1 (RpsA) was repressed; however, the expression of EF-Tu, EF-G and TypA was up-regulated at both protein and transcript levels. This suggests a reduction of protein synthesis in response to acid stress along with possible enhancement of the translational accuracy and protein folding. It is noteworthy that the putative transcriptional regulator Ldb0677 was 1.84-fold up-regulated. Heterologous expression of Ldb0677 was shown to significantly enhance acid resistance in host strain Lactococcus lactis. To clarify its role in transcriptional regulation network, the DNA-binding specificity of Ldb0677 was determined using bacterial one-hybrid and electrophoretic mobility shift assay. The identification of a binding motif (SSTAGACR) present in the promoter regions of 22 genes indicates that it might function as a major regulator in acid stress response in L. bulgaricus. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Anthelmintic metabolism in parasitic helminths: proteomic insights.

    Brophy, Peter M; MacKintosh, Neil; Morphew, Russell M

    2012-08-01

    Anthelmintics are the cornerstone of parasitic helminth control. Surprisingly, understanding of the biochemical pathways used by parasitic helminths to detoxify anthelmintics is fragmented, despite the increasing global threat of anthelmintic resistance within the ruminant and equine industries. Reductionist biochemistry has likely over-estimated the enzymatic role of glutathione transferases in anthelmintic metabolism and neglected the potential role of the cytochrome P-450 superfamily (CYPs). Proteomic technologies offers the opportunity to support genomics, reverse genetics and pharmacokinetics, and provide an integrated insight into both the cellular mechanisms underpinning response to anthelmintics and also the identification of biomarker panels for monitoring the development of anthelmintic resistance. To date, there have been limited attempts to include proteomics in anthelmintic metabolism studies. Optimisations of membrane, post-translational modification and interaction proteomic technologies in helminths are needed to especially study Phase I CYPs and Phase III ABC transporter pumps for anthelmintics and their metabolites.

  16. Advances in Proteomics of Mycobacterium leprae.

    Parkash, O; Singh, B P

    2012-04-01

    Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

  17. The Use of Proteomics in Assisted Reproduction.

    Kosteria, Ioanna; Anagnostopoulos, Athanasios K; Kanaka-Gantenbein, Christina; Chrousos, George P; Tsangaris, George T

    2017-01-01

    Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  18. Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01.

    Lv, Long-Xian; Yan, Ren; Shi, Hai-Yan; Shi, Ding; Fang, Dai-Qiong; Jiang, Hui-Yong; Wu, Wen-Rui; Guo, Fei-Fei; Jiang, Xia-Wei; Gu, Si-Lan; Chen, Yun-Bo; Yao, Jian; Li, Lan-Juan

    2017-01-06

    Lactobacillus salivarius LI01, isolated from healthy humans, has demonstrated probiotic properties in the prevention and treatment of liver failure. Tolerance to bile stress is crucial to allow lactobacilli to survive in the gastrointestinal tract and exert their benefits. In this work, we used a Digital Gene Expression transcriptomic and iTRAQ LC-MS/MS proteomic approach to examine the characteristics of LI01 in response to bile stress. Using culture medium with or without 0.15% ox bile, 591 differentially transcribed genes and 347 differentially expressed proteins were detected in LI01. Overall, we found the bile resistance of LI01 to be based on a highly remodeled cell envelope and a reinforced bile efflux system rather than on the activity of bile salt hydrolases. Additionally, some differentially expressed genes related to regulatory systems, the general stress response and central metabolism processes, also play roles in stress sensing, bile-induced damage prevention and energy efficiency. Moreover, bile salts appear to enhance proteolysis and amino acid uptake (especially aromatic amino acids) by LI01, which may support the liver protection properties of this strain. Altogether, this study establishes a model of global response mechanism to bile stress in L. salivarius LI01. L. salivarius strain LI01 exhibits not only antibacterial and antifungal properties but also exerts a good health-promoting effect in acute liver failure. As a potential probiotic strain, the bile-tolerance trait of strain LI01 is important, though this has not yet been explored. In this study, an analysis based on DGE and iTRAQ was performed to investigate the gene expression in strain LI01 under bile stress at the mRNA and protein levels, respectively. To our knowledge, this work also represents the first combined transcriptomic and proteomic analysis of the bile stress response mechanism in L. salivarius. Copyright © 2016. Published by Elsevier B.V.

  19. iTRAQ Mitoproteome Analysis Reveals Mechanisms of Programmed Cell Death in Arabidopsis thaliana Induced by Ochratoxin A

    Yan Wang

    2017-05-01

    Full Text Available Ochratoxin A (OTA is one of the most common and dangerous mycotoxins in the world. Previous work indicated that OTA could elicit spontaneous HR-like lesions formation Arabidopsis thaliana, reactive oxygen species (ROS play an important role in OTA toxicity, and their major endogenous source is mitochondria. However, there has been no evidence as to whether OTA induces directly PCD in plants until now. In this study, the presence of OTA in Arabidopsis thaliana leaves triggered accelerated respiration, increased production of mitochondrial ROS, the opening of ROS-dependent mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential as well as the release of cytochrome c into the cytosol. There were 42 and 43 significantly differentially expressed proteins identified in response to exposure to OTA for 8 and 24 h, respectively, according to iTRAQ analysis. These proteins were mainly involved in perturbation of the mitochondrial electron transport chain, interfering with ATP synthesis and inducing PCD. Digital gene expression data at transcriptional level was consistent with the cell death induced by OTA being PCD. These results indicated that mitochondrial dysfunction was a prerequisite for OTA-induced PCD and the initiation and execution of PCD via a mitochondrial-mediated pathway.

  20. RNA-Seq and iTRAQ Reveal the Dwarfing Mechanism of Dwarf Polish Wheat (Triticum polonicum L.).

    Wang, Yi; Xiao, Xue; Wang, Xiaolu; Zeng, Jian; Kang, Houyang; Fan, Xing; Sha, Lina; Zhang, Haiqin; Zhou, Yonghong

    2016-01-01

    The dwarfing mechanism of Rht-dp in dwarf Polish wheat (DPW) is unknown. Each internode of DPW was significantly shorter than it in high Polish wheat (HPW), and the dwarfism was insensitive to photoperiod, abscisic acid (ABA), gibberellin (GA), cytokinin (CK), auxin and brassinolide (BR). To understand the mechanism, three sets of transcripts, DPW, HPW, and a chimeric set (a combination of DPW and HPW), were constructed using RNA sequencing (RNA-Seq). Based on the chimeric transcripts, 2,446 proteins were identified using isobaric tags for relative and absolute quantification (iTRAQ). A total of 108 unigenes and 12 proteins were considered as dwarfism-related differentially expressed genes (DEGs) and differentially expressed proteins (DEPs), respectively. Among of these DEGs and DEPs, 6 DEGs and 6 DEPs were found to be involved in flavonoid and S-adenosyl-methionine (SAM) metabolisms; 5 DEGs and 3 DEPs were involved in cellulose metabolism, cell wall plasticity and cell expansion; 2 DEGs were auxin transporters; 2 DEPs were histones; 1 DEP was a peroxidase. These DEGs and DEPs reduced lignin and cellulose contents, increased flavonoid content, possibly decreased S-adenosyl-methionine (SAM) and polyamine contents and increased S-adenosyl-L-homocysteine hydrolase (SAHH) content in DPW stems, which could limit auxin transport and reduce extensibility of the cell wall, finally limited cell expansion (the cell size of DPW was significantly smaller than HPW cells) and caused dwarfism in DPW.

  1. Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization.

    Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo

    2015-01-01

    Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of

  2. Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

    Yanlong Wang

    Full Text Available We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli, Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology and KEGG (Kyoto encyclopedia of genes and genomes enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species scavenging, membrane proteins and ABC (ATP binding cassette transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.

  3. Quantitative proteomic analysis for novel biomarkers of buccal squamous cell carcinoma arising in background of oral submucous fibrosis

    Liu, Wen; Zeng, Lijuan; Li, Ning; Wang, Fei; Jiang, Canhua; Guo, Feng; Chen, Xinqun; Su, Tong; Xu, Chunjiao; Zhang, Shanshan; Fang, Changyun

    2016-01-01

    In South and Southeast Asian, the majority of buccal squamous cell carcinoma (BSCC) can arise from oral submucous fibrosis (OSF). BSCCs develop in OSF that are often not completely resected, causing local relapse. The aim of our study was to find candidate protein biomarkers to detect OSF and predict prognosis in BSCCs by quantitative proteomics approaches. We compared normal oral mucosa (NBM) and paired biopsies of BSCC and OSF by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) to discover proteins with differential expression. Gene Ontology and KEGG networks were analyzed. The prognostic value of biomarkers was evaluated in 94 BSCCs accompanied with OSF. Significant associations were assessed by Kaplan-Meier survival and Cox-proportional hazards analysis. In total 30 proteins were identified with significantly different expression (false discovery rate < 0.05) among three tissues. Two consistently upregulated proteins, ANXA4 and FLNA, were validated. The disease-free survival was negatively associated with the expression of ANXA4 (hazard ratio, 3.4; P = 0.000), FLNA (hazard ratio, 2.1; P = 0.000) and their combination (hazard ratio, 8.8; P = 0.002) in BSCCs. The present study indicates that iTRAQ quantitative proteomics analysis for tissues of BSCC and OSF is a reliable strategy. A significantly up-regulated ANXA4 and FLNA could be not only candidate biomarkers for BSCC prognosis but also potential targets for its therapy. The online version of this article (doi:10.1186/s12885-016-2650-1) contains supplementary material, which is available to authorized users

  4. Proteomic analysis of an environmental isolate of Rhodotorula mucilaginosa after arsenic and cadmium challenge: Identification of a protein expression signature for heavy metal exposure.

    Ilyas, Sidra; Rehman, Abdul; Coelho, Ana Varela; Sheehan, David

    2016-06-01

    A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43±3.34), NaAsO2 (14.76±2.14), CuSO4 (14.73±2.49), and Pb(NO3)2 (15.74±5.3) versus control (7.67±0.95). GSH:GSSG ratio decreased with CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2 and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2. Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast. Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as

  5. Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

    Magro, Massimiliano; Zaccarin, Mattia; Miotto, Giovanni; Da Dalt, Laura; Baratella, Davide; Fariselli, Piero; Gabai, Gianfranco; Vianello, Fabio

    2018-05-01

    Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of a S1 -casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the

  6. Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.

    Sebela, Marek; Rehulka, Pavel; Kábrt, Jaromír; Rehulková, Helena; Ozdian, Tomás; Raus, Martin; Franc, Vojtech; Chmelík, Josef

    2009-11-01

    An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis. Copyright 2009 John Wiley & Sons, Ltd.

  7. Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

    Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Grupp, Kara; Chen, Sixue; Wendel, Jonathan F

    2013-10-01

    Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  8. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  9. Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries.

    Oda, Teiji; Yamaguchi, Akane; Yokoyama, Masao; Shimizu, Koji; Toyota, Kosaku; Nikai, Tetsuro; Matsumoto, Ken-Ichi

    2014-10-01

    Deep hypothermic circulatory arrest (DHCA) is a protective method against brain ischemia in aortic surgery. However, the possible effects of DHCA on the plasma proteins remain to be determined. In the present study, we used novel high‑throughput technology to compare the plasma proteomes during DHCA (22˚C) with selective cerebral perfusion (SCP, n=7) to those during normothermic cardiopulmonary bypass (CPB, n=7). Three plasma samples per patient were obtained during CPB: T1, prior to cooling; T2, during hypothermia; T3, after rewarming for the DHCA group and three corresponding points for the normothermic group. A proteomic analysis was performed using isobaric tag for relative and absolute quantification (iTRAQ) labeling tandem mass spectrometry to assess quantitative protein changes. In total, the analysis identified 262 proteins. The bioinformatics analysis revealed a significant upregulation of complement activation at T2 in normothermic CPB, which was suppressed in DHCA. These findings were confirmed by the changes of the terminal complement complex (SC5b‑9) levels. At T3, however, the level of SC5b‑9 showed a greater increase in DHCA compared to normothermic CPB, while 48 proteins were significantly downregulated in DHCA. The results demonstrated that DHCA and rewarming potentially exert a significant effect on the plasma proteome in patients undergoing aortic surgery.

  10. [Methods of quantitative proteomics].

    Kopylov, A T; Zgoda, V G

    2007-01-01

    In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

  11. In silico proteome analysis to facilitate proteomics experiments using mass spectrometry

    Lindo Micheal

    2003-08-01

    Full Text Available Abstract Proteomics experiments typically involve protein or peptide separation steps coupled to the identification of many hundreds to thousands of peptides by mass spectrometry. Development of methodology and instrumentation in this field is proceeding rapidly, and effective software is needed to link the different stages of proteomic analysis. We have developed an application, proteogest, written in Perl that generates descriptive and statistical analyses of the biophysical properties of multiple (e.g. thousands protein sequences submitted by the user, for instance protein sequences inferred from the complete genome sequence of a model organism. The application also carries out in silico proteolytic digestion of the submitted proteomes, or subsets thereof, and the distribution of biophysical properties of the resulting peptides is presented. proteogest is customizable, the user being able to select many options, for instance the cleavage pattern of the digestion treatment or the presence of modifications to specific amino acid residues. We show how proteogest can be used to compare the proteomes and digested proteome products of model organisms, to examine the added complexity generated by modification of residues, and to facilitate the design of proteomics experiments for optimal representation of component proteins.

  12. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  13. Proteomics Development and Application for Bioforensics

    Wahl, Karen L.; Wunschel, David S.; Clowers, Brian H.

    2010-09-15

    Proteomics is a relatively new scientific discipline dedicated to the comprehensive study of the protein composition of biological systems. While genomic sequencing is an invaluable tool for bioforensic sample identification, proteomics complements genomics in that the genes present in an organism code for the proteins that can be present in a microorganism. Many proteins are conserved for general identification while other protein expression varies with environment/growth state/growth conditions (i.e. not all proteins are expressed at any given time or condition) providing additional information beyond genomic analysis. This expression specificity and the relative stability of proteins with respect to genetic material make them attractive targets for microorganism identification and forensic applications to complement genomic approaches. Proteomic analysis depends upon the availability of genome sequences of the relevant organisms or their near relatives. The known amino acid sequences for potential proteins within the database can be compared to amino acid sequences of actual proteins present in a sample as determined with high mass accuracy by mass spectrometry for identification of the proteins in the sample. With the development of technology for rapid genome sequencing of organisms, the known protein database is growing, supporting improved identification of the proteins present in a sample. Recent developments in mass spectrometry instrumentation and microbial sequencing are leading to an increased growth in application of proteomics to microbiology, pathogen detection, disease diagnosis and microbial forensics as well as other biological disciplines. Mass spectrometry analysis does not require a priori knowledge of the sample or expected targets to gain meaningful.

  14. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

    Annemarie Kramer

    Full Text Available The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

  15. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

    Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

    2015-01-01

    The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

  16. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

    Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F.; Labes, Antje

    2015-01-01

    The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

  17. Proteomic Comparison of Basal Endosperm in Maize miniature1 Mutant and its Wild-type Mn1

    Cecilia eSilva-Sanchez

    2013-06-01

    Full Text Available Developing endosperm in maize seed is a major site for biosynthesis and storage of starch and proteins, and of immense economic importance for its role in food, feed and biofuel production. The basal part of endosperm performs a major role in solute, water and nutrition acquisition from mother plant to sustain these functions. The miniature1 (mn1 mutation is a loss-of-function mutation of the Mn1-encoded cell wall invertase that is entirely expressed in the basal endosperm and is essential for many of the metabolic and signaling functions associated with metabolically released hexose sugars in developing endosperm. Here we report a comparative proteomic study between Mn1 and mn1 basal endosperm to better understand basis of pleiotropic effects on many diverse traits in the mutant. Specifically, we used iTRAQ based quantitative proteomics combined with Gene Ontology and bioinformatics to understand functional basis of the proteomic information. A total of 2518 proteins were identified from soluble and cell wall associated protein fractions; of these 131 proteins were observed to be differentially expressed in the two genotypes. The main functional groups of proteins that were significantly different were those involved in the carbohydrate metabolic and catabolic process, and cell homeostasis. The study constitutes the first proteomic analysis of basal endosperm cell layers in relation to endosperm growth and development in maize.

  18. Top Down proteomics: Facts and perspectives

    Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L., E-mail: n-kelleher@northwestern.edu

    2014-03-21

    Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

  19. Top Down proteomics: Facts and perspectives

    Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L.

    2014-01-01

    Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years

  20. Comparative proteomics analysis of placenta from pregnant women with intrahepatic cholestasis of pregnancy.

    Zhang, Ting; Guo, Yueshuai; Guo, Xuejiang; Zhou, Tao; Chen, Daozhen; Xiang, Jingying; Zhou, Zuomin

    2013-01-01

    Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach. The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay. Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased. This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.

  1. Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

    Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

    2016-01-01

    Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

  2. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    An, Xiaomeng

    2017-06-21

    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

  3. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Qingzhu Hua

    2016-09-01

    Full Text Available Red dragon fruit or red pitaya (Hylocereus polyrhizus is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  4. Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach

    Daniel V. Oliveira

    2012-01-01

    Full Text Available Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM. We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2 were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

  5. Proteomic Analysis of Chicken Skeletal Muscle during Embryonic Development

    Hongjia Ouyang

    2017-05-01

    Full Text Available Embryonic growth and development of skeletal muscle is a major determinant of muscle mass, and has a significant effect on meat production in chicken. To assess the protein expression profiles during embryonic skeletal muscle development, we performed a proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ in leg muscle tissues of female Xinghua chicken at embryonic age (E 11, E16, and 1-day post hatch (D1. We identified 3,240 proteins in chicken embryonic muscle and 491 of them were differentially expressed (fold change ≥ 1.5 or ≤ 0.666 and p < 0.05. There were 19 up- and 32 down-regulated proteins in E11 vs. E16 group, 238 up- and 227 down-regulated proteins in E11 vs. D1 group, and 13 up- and 5 down-regulated proteins in E16 vs. D1 group. Protein interaction network analyses indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis, muscle contraction, and oxidative phosphorylation. Integrative analysis of proteome and our previous transcriptome data found 189 differentially expressed proteins that correlated with their mRNA level. The interactions between these proteins were also involved in muscle contraction and oxidative phosphorylation pathways. The lncRNA-protein interaction network found four proteins DMD, MYL3, TNNI2, and TNNT3 that are all involved in muscle contraction and may be lncRNA regulated. These results provide several candidate genes for further investigation into the molecular mechanisms of chicken embryonic muscle development, and enable us to better understanding their regulation networks and biochemical pathways.

  6. Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity.

    Stolle, Theresa; Grondinger, Freya; Dunkel, Andreas; Meng, Chen; Médard, Guillaume; Kuster, Bernhard; Hofmann, Thomas

    2017-10-25

    To investigate the role of perireceptor events in inter-individual variability in salt taste sensitivity, 31 volunteers were monitored in their detection functions for sodium chloride (NaCl) and classified into sensitive (0.6-1.7 mmol/L), medium-sensitive (1.8-6.9 mmol/L), and nonsensitive (7.0-11.2 mmol/L) subjects. Chemosensory intervention of NaCl-sensitive (S + ) and nonsensitive (S - ) panellists with potassium chloride, ammonium chloride, and sodium gluconate showed the salt taste sensitivity to be specific for NaCl. As no significant differences were found between S + and S - subjects in salivary sodium and protein content, salivary proteome differences and their stimulus-induced dynamic changes were analyzed by tryptic digestion, iTRAQ labeling, and liquid chromatography-tandem mass spectrometry analysis. Differences in the salivary proteome between S + and S - subjects were found primarily in resting saliva and were largely independent of the dynamic alterations observed upon salt stimulation. Gene ontology enrichment analysis of key proteins, i.e., immunoglobulin heavy constant y1, myeloblastin, cathepsin G, and kallikrein, revealed significantly increased serine-type endopeptidase activity for the S + group, while the S - group exhibited augmented cysteine-type endopeptidase inhibitor activity by increased abundances in lipocalin-1 and cystatin-D, -S, and -SN, respectively. As proteases have been suggested to facilitate transepithelial sodium transport by cleaving the y-subunit of the epithelial sodium channel (ENaC) and protease inhibitors have been shown to reduce ENaC-mediated sodium transport, the differentially modulated proteolytic activity patterns observed in vivo for S + and S - subjects show evidence of them playing a crucial role in affecting human NaCl sensitivity.

  7. Proteomics of Trypanosoma evansi infection in rodents.

    Roy, Nainita; Nageshan, Rishi Kumar; Pallavi, Rani; Chakravarthy, Harshini; Chandran, Syama; Kumar, Rajender; Gupta, Ashok Kumar; Singh, Raj Kumar; Yadav, Suresh Chandra; Tatu, Utpal

    2010-03-22

    Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS). Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more. Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the

  8. Proteomics of Trypanosoma evansi infection in rodents.

    Nainita Roy

    2010-03-01

    Full Text Available Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS.Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a

  9. ProteomicsDB.

    Schmidt, Tobias; Samaras, Patroklos; Frejno, Martin; Gessulat, Siegfried; Barnert, Maximilian; Kienegger, Harald; Krcmar, Helmut; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Kuster, Bernhard; Wilhelm, Mathias

    2018-01-04

    ProteomicsDB (https://www.ProteomicsDB.org) is a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ProteomicsDB was first released in 2014 to enable the interactive exploration of the first draft of the human proteome. To date, it contains quantitative data from 78 projects totalling over 19k LC-MS/MS experiments. A standardized analysis pipeline enables comparisons between multiple datasets to facilitate the exploration of protein expression across hundreds of tissues, body fluids and cell lines. We recently extended the data model to enable the storage and integrated visualization of other quantitative omics data. This includes transcriptomics data from e.g. NCBI GEO, protein-protein interaction information from STRING, functional annotations from KEGG, drug-sensitivity/selectivity data from several public sources and reference mass spectra from the ProteomeTools project. The extended functionality transforms ProteomicsDB into a multi-purpose resource connecting quantification and meta-data for each protein. The rich user interface helps researchers to navigate all data sources in either a protein-centric or multi-protein-centric manner. Several options are available to download data manually, while our application programming interface enables accessing quantitative data systematically. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-05-19

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

  11. Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

    Sabine Matallana-Surget

    Full Text Available The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

  12. Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project.

    Omenn, Gilbert S; Lane, Lydie; Lundberg, Emma K; Overall, Christopher M; Deutsch, Eric W

    2017-12-01

    The Human Proteome Organization (HUPO) Human Proteome Project (HPP) continues to make progress on its two overall goals: (1) completing the protein parts list, with an annual update of the HUPO draft human proteome, and (2) making proteomics an integrated complement to genomics and transcriptomics throughout biomedical and life sciences research. neXtProt version 2017-01-23 has 17 008 confident protein identifications (Protein Existence [PE] level 1) that are compliant with the HPP Guidelines v2.1 ( https://hupo.org/Guidelines ), up from 13 664 in 2012-12 and 16 518 in 2016-04. Remaining to be found by mass spectrometry and other methods are 2579 "missing proteins" (PE2+3+4), down from 2949 in 2016. PeptideAtlas 2017-01 has 15 173 canonical proteins, accounting for nearly all of the 15 290 PE1 proteins based on MS data. These resources have extensive data on PTMs, single amino acid variants, and splice isoforms. The Human Protein Atlas v16 has 10 492 highly curated protein entries with tissue and subcellular spatial localization of proteins and transcript expression. Organ-specific popular protein lists have been generated for broad use in quantitative targeted proteomics using SRM-MS or DIA-SWATH-MS studies of biology and disease.

  13. Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics.

    Kristian E Swearingen

    2016-04-01

    Full Text Available Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP, conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.

  14. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

    Preeti Shahi

    2016-01-01

    Full Text Available Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

  15. Data for iTRAQ-based quantitative proteomics analysis of Brassica napus leaves in response to chlorophyll deficiency

    Pu Chu

    2015-03-01

    Full Text Available The essential pigment chlorophyll (Chl plays important roles in light harvesting and energy transfer during photosynthesis. Here we present the data from a comparative proteomic analysis of chlorophyll-deficient Brassica napus mutant cde1 and its corresponding wild-type using the iTRAQ approach (Pu Chu et al., 2014 [1]. The distribution of length and number of peptides, mass and sequence coverage of proteins identified was calculated, and the repeatability of the replicates was analyzed. A total of 443 differentially expressed proteins were identified in B. napus leaves, including 228 down-accumulated proteins mainly involved in photosynthesis, porphyrin and chlorophyll metabolism, biosynthesis of secondary metabolites, carbon fixation and 215 up-accumulated proteins that enriched in the spliceosome, mRNA surveillance and RNA degradation.

  16. Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome.

    Aragonès, Gemma; Auguet, Teresa; Guiu-Jurado, Esther; Berlanga, Alba; Curriu, Marta; Martinez, Salomé; Alibalic, Ajla; Aguilar, Carmen; Hernández, Esteban; Camara, María-Luisa; Canela, Núria; Herrero, Pol; Ruyra, Xavier; Martín-Paredero, Vicente; Richart, Cristóbal

    2016-03-04

    Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

  17. Proteomics in the investigation of HIV-1 interactions with host proteins.

    Li, Ming

    2015-02-01

    Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Genomics and proteomics: Applications in autoimmune diseases

    Wolfgang Hueber

    2009-08-01

    Full Text Available Wolfgang Hueber1,2,3, William H Robinson1,21VA Palo Alto Health Care System, Palo Alto, CA, USA; 2Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA; 3Novartis Institutes of Biomedical Research, Novartis, Basle, SwitzerlandAbstract: Tremendous progress has been made over the past decade in the development and refinement of genomic and proteomic technologies for the identification of novel drug targets and molecular signatures associated with clinically important disease states, disease subsets, or differential responses to therapies. The rapid progress in high-throughput technologies has been preceded and paralleled by the elucidation of cytokine networks, followed by the stepwise clinical development of pathway-specific biological therapies that revolutionized the treatment of autoimmune diseases. Together, these advances provide opportunities for a long-anticipated personalized medicine approach to the treatment of autoimmune disease. The ever-increasing numbers of novel, innovative therapies will need to be harnessed wisely to achieve optimal long-term outcomes in as many patients as possible while complying with the demands of health authorities and health care providers for evidence-based, economically sound prescription of these expensive drugs. Genomic and proteomic profiling of patients with autoimmune diseases holds great promise in two major clinical areas: (1 rapid identification of new targets for the development of innovative therapies and (2 identification of patients who will experience optimal benefit and minimal risk from a specific (targeted therapy. In this review, we attempt to capture important recent developments in the application of genomic and proteomic technologies to translational research by discussing informative examples covering a diversity of autoimmune diseases.Keywords: proteomics, genomics, autoimmune diseases, antigen microarrays, 2-Dih, rheumatoid arthritis

  19. Systems toxicology: applications of toxicogenomics, transcriptomics, proteomics and metabolomics in toxicology

    Heijne, W.H.M.; Kienhuis, A.S.; Ommen, van B.; Stierum, R.; Groten, J.P.

    2005-01-01

    Toxicogenomics can facilitate the identification and characterization of toxicity, as illustrated in this review. Toxicogenomics, the application of the functional genomics technologies (transcriptomics, proteomics and metabolomics) in toxicology enables the study of adverse effects of xenobiotic

  20. Mining the active proteome of Arabidopsis thaliana

    Renier A. L. Van Der Hoorn

    2011-11-01

    Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

  1. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

    Aniuska Becerra-Artiles

    Full Text Available Most of humanity is chronically infected with human herpesvirus 6 (HHV-6, with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48 and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune

  2. Proteome Modification in Tomato Plants upon Long-Term Aluminum Treatment.

    Zhou, Suping; Okekeogbu, Ikenna; Sangireddy, Sasikiran; Ye, Zhujia; Li, Hui; Bhatti, Sarabjit; Hui, Dafeng; McDonald, Daniel W; Yang, Yong; Giri, Shree; Howe, Kevin J; Fish, Tara; Thannhauser, Theodore W

    2016-05-06

    This study aimed to identify the aluminum (Al)-induced proteomes in tomato (Solanum lycopersicum, "Micro-Tom") after long-term exposure to the stress factor. Plants were treated in Magnavaca's solution (pH 4.5) supplemented with 7.5 μM Al(3+) ion activity over a 4 month period beginning at the emergence of flower buds and ending when the lower mature leaves started to turn yellow. Proteomes were identified using a 8-plex isobaric tags for relative and absolute quantification (iTRAQ) labeling strategy followed by a two-dimensional (high- and low-pH) chromatographic separation and final generation of tandem mass spectrometry (MS/MS) spectra of tryptic peptides on an LTQ-Orbitrap Elite mass spectrometer. Principal component analysis revealed that the Al-treatment had induced systemic alterations in the proteomes from roots and leaves but not seed tissues. The significantly changed root proteins were shown to have putative functions in Al(3+) ion uptake and transportation, root development, and a multitude of other cellular processes. Changes in the leaf proteome indicate that the light reaction centers of photosynthetic machinery are the primary targets of Al-induced stress. Embryo and seed-coat tissues derived from Al-treated plants were enriched with stress proteins. The biological processes involving these Al-induced proteins concur with the physiological and morphological changes, such as the disturbance of mineral homeostasis (higher contents of Al, P, and Fe and reduced contents of S, Zn, and Mn in Al-treated compared to nontreated plants) in roots and smaller sizes of roots and the whole plants. More importantly, the identified significant proteins might represent a molecular mechanism for plants to develop toward establishing the Al tolerance and adaptation mechanism over a long period of stress treatment.

  3. Transcriptomic and proteomic analysis of Oenococcus oeni adaptation to wine stress conditions

    Mar Margalef-Català

    2016-09-01

    Full Text Available Oenococcus oeni, the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to

  4. Differential proteomic analysis reveals sequential heat stress-responsive regulatory network in radish (Raphanus sativus L.) taproot.

    Wang, Ronghua; Mei, Yi; Xu, Liang; Zhu, Xianwen; Wang, Yan; Guo, Jun; Liu, Liwang

    2018-05-01

    Differential abundance protein species (DAPS) involved in reducing damage and enhancing thermotolerance in radish were firstly identified. Proteomic analysis and omics association analysis revealed a HS-responsive regulatory network in radish. Heat stress (HS) is a major destructive factor influencing radish production and supply in summer, for radish is a cool season vegetable crop being susceptible to high temperature. In this study, the proteome changes of radish taproots under 40 °C treatment at 0 h (Control), 12 h (Heat12) and 24 h (Heat24) were analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) approach. In total, 2258 DAPS representing 1542 differentially accumulated uniprotein species which respond to HS were identified. A total of 604, 910 and 744 DAPS was detected in comparison of Control vs. Heat12, Control vs. Heat24, and Heat12 vs. Heat24, respectively. Gene ontology and pathway analysis showed that annexin, ubiquitin-conjugating enzyme, ATP synthase, heat shock protein (HSP) and other stress-related proteins were predominately enriched in signal transduction, stress and defense pathways, photosynthesis and energy metabolic pathways, working cooperatively to reduce stress-induced damage in radish. Based on iTRAQ combined with the transcriptomics analysis, a schematic model of a sequential HS-responsive regulatory network was proposed. The initial sensing of HS occurred at the plasma membrane, and then key components of stress signal transduction triggered heat-responsive genes in the plant protective metabolism to re-establish homeostasis and enhance thermotolerance. These results provide new insights into characteristics of HS-responsive DAPS and facilitate dissecting the molecular mechanisms underlying heat tolerance in radish and other root crops.

  5. iTRAQ-Based Proteomic Analysis of Sublethally Injured Escherichia coli O157:H7 Cells Induced by High Pressure Carbon Dioxide

    Xiufang Bi

    2017-12-01

    Full Text Available High pressure carbon dioxide (HPCD could cause sublethally injured cells (SICs, which may cause food poisoning and spoilage during food storage and limit its application. Therefore, the formation of SICs of Escherichia coli O157:H7 was investigated by isobaric tag for relative and absolute quantification (iTRAQ proteomic methods in this study for better controlling the SICs induced by HPCD. A total of 2,446 proteins was identified by iTRAQ, of which 93 and 29 were significantly differentially expressed in the SICs compared with live control cells (CKL and dead control cells (CKD, respectively. Among the 93 differentially expressed proteins (DEP in the SICs compared with CKL, 65 proteins showed down-regulation and 28 showed up-regulation. According to the comprehensive proteome coverage analysis, the SICs survived under HPCD by reducing carbohydrate decomposing, lipid transport and metabolism, amino acid transport and metabolism, transcription and translation, DNA replication and repair. Besides, the SICs showed stress response, DNA damage response and an increased carbohydrate transport, peptidoglycan synthesis and disulfide bond formation to HPCD. Among the 29 DEP in the SICs compared with CKD, 12 proteins showed down-regulation and 17 showed up-regulation. According to the comprehensive proteome coverage analysis, the SICs survived under HPCD by accumulation of cell protective agents like carbohydrates and amino acids, and decreasing transcription and translation activities. Results showed that the formation of the SICs with low metabolic activity and high survival ability was a survival strategy for E. coli O157:H7 against HPCD.

  6. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

    Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wisniewski, Jacek R; Jun, Wang; Mann, Matthias

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

  7. A novel multidimensional protein identification technology approach combining protein size exclusion prefractionation, peptide zwitterion-ion hydrophilic interaction chromatography, and nano-ultraperformance RP chromatography/nESI-MS2 for the in-depth analysis of the serum proteome and phosphoproteome: application to clinical sera derived from humans with benign prostate hyperplasia.

    Garbis, Spiros D; Roumeliotis, Theodoros I; Tyritzis, Stavros I; Zorpas, Kostas M; Pavlakis, Kitty; Constantinides, Constantinos A

    2011-02-01

    The current proof-of-principle study was aimed toward development of a novel multidimensional protein identification technology (MudPIT) approach for the in-depth proteome analysis of human serum derived from patients with benign prostate hyperplasia (BPH) using rational chromatographic design principles. This study constituted an extension of our published work relating to the identification and relative quantification of potential clinical biomarkers in BPH and prostate cancer (PCa) tissue specimens. The proposed MudPIT approach encompassed the use of three distinct yet complementary liquid chromatographic chemistries. High-pressure size-exclusion chromatography (SEC) was used for the prefractionation of serum proteins followed by their dialysis exchange and solution phase trypsin proteolysis. The tryptic peptides were then subjected to offline zwitterion-ion hydrophilic interaction chromatography (ZIC-HILIC) fractionation followed by their online analysis with reversed-phase nano-ultraperformance chromatography (RP-nUPLC) hyphenated to nanoelectrospray ionization-tandem mass spectrometry using an ion trap mass analyzer. For the spectral processing, the sequential use of the SpectrumMill, Scaffold, and InsPecT software tools was applied for the tryptic peptide product ion MS(2) spectral processing, false discovery rate (FDR) assessment, validation, and protein identification. This milestone serum analysis study allowed the confident identification of over 1955 proteins (p ≤ 0.05; FDR ≤ 5%) with a broad spectrum of biological and physicochemical properties including secreted, tissue-specific proteins spanning approximately 12 orders of magnitude as they occur in their native abundance levels in the serum matrix. Also encompassed in this proteome was the confident identification of 375 phosphoproteins (p ≤ 0.05; FDR ≤ 5%) with potential importance to cancer biology. To demonstrate the performance characteristics of this novel MudPIT approach, a comparison

  8. Identification of a novel biomarker candidate, a 4.8-kDa peptide fragment from a neurosecretory protein VGF precursor, by proteomic analysis of cerebrospinal fluid from children with acute encephalopathy using SELDI-TOF-MS

    Fujino Osamu

    2011-08-01

    Full Text Available Abstract Background Acute encephalopathy includes rapid deterioration and has a poor prognosis. Early intervention is essential to prevent progression of the disease and subsequent neurologic complications. However, in the acute period, true encephalopathy cannot easily be differentiated from febrile seizures, especially febrile seizures of the complex type. Thus, an early diagnostic marker has been sought in order to enable early intervention. The purpose of this study was to identify a novel marker candidate protein differentially expressed in the cerebrospinal fluid (CSF of children with encephalopathy using proteomic analysis. Methods For detection of biomarkers, CSF samples were obtained from 13 children with acute encephalopathy and 42 children with febrile seizure. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS technology, which is currently applied in many fields of biological and medical sciences. Diagnosis was made by at least two pediatric neurologists based on the clinical findings and routine examinations. All specimens were collected for diagnostic tests and the remaining portion of the specimens were used for the SELDI-TOF MS investigations. Results In experiment 1, CSF from patients with febrile seizures (n = 28, patients with encephalopathy (n = 8 (including influenza encephalopathy (n = 3, encephalopathy due to rotavirus (n = 1, human herpes virus 6 (n = 1 were used for the SELDI analysis. In experiment 2, SELDI analysis was performed on CSF from a second set of febrile seizure patients (n = 14 and encephalopathy patients (n = 5. We found that the peak with an m/z of 4810 contributed the most to the separation of the two groups. After purification and identification of the 4.8-kDa protein, a 4.8-kDa proteolytic peptide fragment from the neurosecretory protein VGF precursor (VGF4.8 was identified as a novel biomarker for encephalopathy. Conclusions

  9. Application of Proteomics and Peptidomics to COPD

    Girolamo Pelaia

    2014-01-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is a complex disorder involving both airways and lung parenchyma, usually associated with progressive and poorly reversible airflow limitation. In order to better characterize the phenotypic heterogeneity and the prognosis of patients with COPD, there is currently an urgent need for discovery and validation of reliable disease biomarkers. Within this context, proteomic and peptidomic techniques are emerging as very valuable tools that can be applied to both systemic and pulmonary samples, including peripheral blood, induced sputum, exhaled breath condensate, bronchoalveolar lavage fluid, and lung tissues. Identification of COPD biomarkers by means of proteomic and peptidomic approaches can thus also lead to discovery of new molecular targets potentially useful to improve and personalize the therapeutic management of this widespread respiratory disease.

  10. Comprehensive data analysis of human ureter proteome

    Sameh Magdeldin

    2016-03-01

    Full Text Available Comprehensive human ureter proteome dataset was generated from OFFGel fractionated ureter samples. Our result showed that among 2217 non-redundant ureter proteins, 751 protein candidates (33.8% were detected in urine as urinary protein/polypeptide or exosomal protein. On the other hand, comparing ureter protein hits (48 that are not shown in corresponding databases to urinary bladder and prostate human protein atlas databases pinpointed 21 proteins that might be unique to ureter tissue. In conclusion, this finding offers future perspectives for possible identification of ureter disease-associated biomarkers such as ureter carcinoma. In addition, Cytoscape GO annotation was examined on the final ureter dataset to better understand proteins molecular function, biological processes, and cellular component. The ureter proteomic dataset published in this article will provide a valuable resource for researchers working in the field of urology and urine biomarker discovery.

  11. Proteomic maps of breast cancer subtypes

    Tyanova, Stefka; Albrechtsen, Reidar; Kronqvist, Pauliina

    2016-01-01

    Systems-wide profiling of breast cancer has almost always entailed RNA and DNA analysis by microarray and sequencing techniques. Marked developments in proteomic technologies now enable very deep profiling of clinical samples, with high identification and quantification accuracy. We analysed 40...... oestrogen receptor positive (luminal), Her2 positive and triple negative breast tumours and reached a quantitative depth of >10,000 proteins. These proteomic profiles identified functional differences between breast cancer subtypes, related to energy metabolism, cell growth, mRNA translation and cell......-cell communication. Furthermore, we derived a signature of 19 proteins, which differ between the breast cancer subtypes, through support vector machine (SVM)-based classification and feature selection. Remarkably, only three proteins of the signature were associated with gene copy number variations and eleven were...

  12. An efficient proteomic approach to analyze agriculture crop biomass

    Flodrová, Dana; Bobálová, Janette

    2013-01-01

    Roč. 32, č. 5 (2013), s. 365-372 ISSN 1572-3887 R&D Projects: GA MŠk 1M0570 Institutional support: RVO:68081715 Keywords : MALDI * biomass * proteomics * identification * hemicellulases Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.039, year: 2013

  13. Sample handling for mass spectrometric proteomic investigations of human sera.

    West-Nielsen, M.; Hogdall, E.V.; Marchiori, E.; Hogdall, C.K.; Schou, C.; Heegaard, N.H.H.

    2005-01-01

    Proteomic investigations of sera are potentially of value for diagnosis, prognosis, choice of therapy, and disease activity assessment by virtue of discovering new biomarkers and biomarker patterns. Much debate focuses on the biological relevance and the need for identification of such biomarkers

  14. The Spectra Count Label-free Quantitation in Cancer Proteomics

    Zhou, Weidong; Liotta, Lance A.; Petricoin, Emanuel F.

    2012-01-01

    Mass spectrometry is used routinely for large-scale protein identification from complex biological mixtures. Recently, relative quantitation approach on the basis of spectra count has been applied in several cancer proteomic studies. In this review, we examine the mechanism of this technique and highlight several important parameters associated with its application.

  15. Changes in protein abundance between tender and tough meat from bovine Longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis

    Bjarnadóttir, S G; Hollung, K; Høy, M

    2012-01-01

    The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4...

  16. Proteomics - new analytical approaches

    Hancock, W.S.

    2001-01-01

    Full text: Recent developments in the sequencing of the human genome have indicated that the number of coding gene sequences may be as few as 30,000. It is clear, however, that the complexity of the human species is dependent on the much greater diversity of the corresponding protein complement. Estimates of the diversity (discrete protein species) of the human proteome range from 200,000 to 300,000 at the lower end to 2,000,000 to 3,000,000 at the high end. In addition, proteomics (the study of the protein complement to the genome) has been subdivided into two main approaches. Global proteomics refers to a high throughput examination of the full protein set present in a cell under a given environmental condition. Focused proteomics refers to a more detailed study of a restricted set of proteins that are related to a specified biochemical pathway or subcellular structure. While many of the advances in proteomics will be based on the sequencing of the human genome, de novo characterization of protein microheterogeneity (glycosylation, phosphorylation and sulfation as well as the incorporation of lipid components) will be required in disease studies. To characterize these modifications it is necessary to digest the protein mixture with an enzyme to produce the corresponding mixture of peptides. In a process analogous to sequencing of the genome, shot-gun sequencing of the proteome is based on the characterization of the key fragments produced by such a digest. Thus, a glycopeptide and hence a specific glycosylation motif will be identified by a unique mass and then a diagnostic MS/MS spectrum. Mass spectrometry will be the preferred detector in these applications because of the unparalleled information content provided by one or more dimensions of mass measurement. In addition, highly efficient separation processes are an absolute requirement for advanced proteomic studies. For example, a combination of the orthogonal approaches, HPLC and HPCE, can be very powerful

  17. Proteomic analysis of the Theileria annulata schizont

    Witschi, M.; Xia, D.; Sanderson, S.; Baumgartner, M.; Wastling, J.M.; Dobbelaere, D.A.E.

    2013-01-01

    The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites. PMID:23178997

  18. Positional proteomics in the era of the human proteome project on the doorstep of precision medicine.

    Eckhard, Ulrich; Marino, Giada; Butler, Georgina S; Overall, Christopher M

    2016-03-01

    Proteolytic processing is a pervasive and irreversible post-translational modification that expands the protein universe by generating new proteoforms (protein isoforms). Unlike signal peptide or prodomain removal, protease-generated proteoforms can rarely be predicted from gene sequences. Positional proteomic techniques that enrich for N- or C-terminal peptides from proteomes are indispensable for a comprehensive understanding of a protein's function in biological environments since protease cleavage frequently results in altered protein activity and localization. Proteases often process other proteases and protease inhibitors which perturbs proteolytic networks and potentiates the initial cleavage event to affect other molecular networks and cellular processes in physiological and pathological conditions. This review is aimed at researchers with a keen interest in state of the art systems level positional proteomic approaches that: (i) enable the study of complex protease-protease, protease-inhibitor and protease-substrate crosstalk and networks; (ii) allow the identification of proteolytic signatures as candidate disease biomarkers; and (iii) are expected to fill the Human Proteome Project missing proteins gap. We predict that these methodologies will be an integral part of emerging precision medicine initiatives that aim to customize healthcare, converting reactive medicine into a personalized and proactive approach, improving clinical care and maximizing patient health and wellbeing, while decreasing health costs by eliminating ineffective therapies, trial-and-error prescribing, and adverse drug effects. Such initiatives require quantitative and functional proteome profiling and dynamic disease biomarkers in addition to current pharmacogenomics approaches. With proteases at the pathogenic center of many diseases, high-throughput protein termini identification techniques such as TAILS (Terminal Amine Isotopic Labeling of Substrates) and COFRADIC (COmbined

  19. Translational plant proteomics: A perspective

    Agrawal, G.K.; Pedreschi, R.; Barkla, B.J.; Bindschedler, L.V.; Cramer, R.; Sarkar, A.; Renaut, J.; Job, D.; Rakwal, R.

    2012-01-01

    Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic

  20. A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus

    Owens, Rebecca A.; Hammel, Stephen; Sheridan, Kevin J.; Jones, Gary W.; Doyle, Sean

    2014-01-01

    A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (pproteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism. PMID:25198175

  1. Dentistry proteomics: from laboratory development to clinical practice.

    Rezende, Taia M B; Lima, Stella M F; Petriz, Bernardo A; Silva, Osmar N; Freire, Mirna S; Franco, Octávio L

    2013-12-01

    Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease. Copyright © 2013 Wiley Periodicals, Inc.

  2. Proteomics in uveal melanoma.

    Ramasamy, Pathma

    2014-01-01

    Uveal melanoma is the most common primary intraocular malignancy in adults, with an incidence of 5-7 per million per year. It is associated with the development of metastasis in about 50% of cases, and 40% of patients with uveal melanoma die of metastatic disease despite successful treatment of the primary tumour. The survival rates at 5, 10 and 15 years are 65%, 50% and 45% respectively. Unlike progress made in many other areas of cancer, uveal melanoma is still poorly understood and survival rates have remained similar over the past 25 years. Recently, advances made in molecular genetics have improved our understanding of this disease and stratification of patients into low risk and high risk for developing metastasis. However, only a limited number of studies have been performed using proteomic methods. This review will give an overview of various proteomic technologies currently employed in life sciences research, and discuss proteomic studies of uveal melanoma.

  3. Establishing Substantial Equivalence: Proteomics

    Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

    Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

  4. Detection of ROS Induced Proteomic Signatures by Mass Spectrometry

    Brian McDonagh

    2017-07-01

    Full Text Available Reversible and irreversible post-translational modifications (PTMs induced by endogenously generated reactive oxygen species (ROS in regulatory enzymes and proteins plays an essential role in cellular signaling. Almost all cellular processes including metabolism, transcription, translation and degradation have been identified as containing redox regulated proteins. Specific redox modifications of key amino acids generated by ROS offers a dynamic and versatile means to rapidly alter the activity or functional structure of proteins in response to biochemical, environmental, genetic and pathological perturbations. How the proteome responds to these stimuli is of critical importance in oxidant physiology, as it can regulate the cell stress response by reversible and irreversible PTMs, affecting protein activity and protein-protein interactions. Due to the highly labile nature of many ROS species, applying redox proteomics can provide a signature footprint of the ROS species generated. Ideally redox proteomic approaches would allow; (1 the identification of the specific PTM, (2 identification of the amino acid residue that is modified and (3 the percentage of the protein containing the PTM. New developments in MS offer the opportunity of a more sensitive targeted proteomic approach and retrospective data analysis. Subsequent bioinformatics analysis can provide an insight into the biochemical and physiological pathways or cell signaling cascades that are affected by ROS generation. This mini-review will detail current redox proteomic approaches to identify and quantify ROS induced PTMs and the subsequent effects on cellular signaling.

  5. Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques

    Ohlendieck, Kay

    2011-02-01

    Abstract Background Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Results Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins. Conclusions This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates.

  6. Data from proteome analysis of Lasiodiplodia theobromae (Botryosphaeriaceae

    Carla C. Uranga

    2017-08-01

    Full Text Available Trunk disease fungi are a global problem affecting many economically important fruiting trees. The Botryosphaeriaceae are a family of trunk disease fungi that require detailed biochemical characterization in order to gain insight into their pathogenicity. The application of a modified Folch extraction to protein extraction from the Botryosphaeriaceae Lasiodiplodia theobromae generated an unprecedented data set of protein identifications from fragmentation analysis and de novo peptide sequencing of its proteome. This article contains data from protein identifications obtained from a database-dependent fragmentation analysis using three different proteomics algorithms (MSGF, Comet and X! Tandem via the SearchGUI proteomics pipeline program and de novo peptide sequencing. Included are data sets of gene ontology annotations using an all-Uniprot ontology database, as well as a Saccharomyces cerevisiae-only and a Candida albicans-only ontology database, in order to discern between those proteins involved in common functions with S. cerevisiae and those in common with the pathogenic yeast C. albicans. Our results reveal the proteome of L. theobromae contains more ontological categories in common to C. albicans, yet possesses a much wider metabolic repertoire than any of the yeasts studied in this work. Many novel proteins of interest were identified for further biochemical characterization and annotation efforts, as further discussed in the article referencing this article (1. Interactive Cytoscape networks of molecular functions of identified peptides using an all-Uniprot ontological database are included. Data, including raw data, are available via ProteomeXchange with identifier PXD005283.

  7. Morphologic identification and proteomic analysis in adult ...

    In gel separation of the resulting supernatant was achieved using1 dimesnsional Sodium dodecyl sulfate polyacrylamide gel electrophoresis (1DE SDS-PAGE). The raw images of the gel were captured using and analyses on 1D Totallabsoft ware. The software identified 7 (seven) protein bands for male and 10 (ten) protein ...

  8. The Redox Proteome*

    Go, Young-Mi; Jones, Dean P.

    2013-01-01

    The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges. PMID:23861437

  9. Pre-fractionation strategies to resolve pea (Pisum sativum sub-proteomes

    Claudia Nicole Meisrimler

    2015-10-01

    Full Text Available Legumes are important crop plants and pea (Pisum sativum L. has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula G. allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins. Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

  10. Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

    Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

    2016-08-11

    Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating

  11. Translational plant proteomics: a perspective.

    Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

    2012-08-03

    Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders.

    Cehofski, Lasse Jørgensen; Honoré, Bent; Vorum, Henrik

    2017-04-28

    Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions.

  13. Proteomics with Mass Spectrometry Imaging: Beyond Amyloid Typing.

    Lavatelli, Francesca; Merlini, Giampaolo

    2018-04-01

    Detection and typing of amyloid deposits in tissues are two crucial steps in the management of systemic amyloidoses. The presence of amyloid deposits is routinely evaluated through Congo red staining, whereas proteomics is now a mainstay in the identification of the deposited proteins. In article number 1700236, Winter et al. [Proteomics 2017, 17, Issue 22] describe a novel method based on MALDI-MS imaging coupled to ion mobility separation and peptide filtering, to detect the presence of amyloid in histology samples and to identify its composition, while preserving the spatial distribution of proteins in tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Proteomic profile of the plant-pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph.

    Pang, Zhili; Chen, Lei; Miao, Jianqiang; Wang, Zhiwen; Bulone, Vincent; Liu, Xili

    2015-09-01

    Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant-pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild-type P. capsici isolate (Hd3) and two pyrimorph-resistant mutants (R3-1 and R3-2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3-1, and R3-2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d-[U-(14) C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Plasma Proteome Biomarkers of Inflammation in School Aged Children in Nepal.

    Sun Eun Lee

    Full Text Available Inflammation is a condition stemming from complex host defense and tissue repair mechanisms, often simply characterized by plasma levels of a single acute reactant. We attempted to identify candidate biomarkers of systemic inflammation within the plasma proteome. We applied quantitative proteomics using isobaric mass tags (iTRAQ tandem mass spectrometry to quantify proteins in plasma of 500 Nepalese children 6-8 years of age. We evaluated those that co-vary with inflammation, indexed by α-1-acid glycoprotein (AGP, a conventional biomarker of inflammation in population studies. Among 982 proteins quantified in >10% of samples, 99 were strongly associated with AGP at a family-wise error rate of 0.1%. Magnitude and significance of association varied more among proteins positively (n = 41 than negatively associated (n = 58 with AGP. The former included known positive acute phase proteins including C-reactive protein, serum amyloid A, complement components, protease inhibitors, transport proteins with anti-oxidative activity, and numerous unexpected intracellular signaling molecules. Negatively associated proteins exhibited distinct differences in abundance between secretory hepatic proteins involved in transporting or binding lipids, micronutrients (vitamin A and calcium, growth factors and sex hormones, and proteins of largely extra-hepatic origin involved in the formation and metabolic regulation of extracellular matrix. With the same analytical approach and the significance threshold, seventy-two out of the 99 proteins were commonly associated with CRP, an established biomarker of inflammation, suggesting the validity of the identified proteins. Our findings have revealed a vast plasma proteome within a free-living population of children that comprise functional biomarkers of homeostatic and induced host defense, nutrient metabolism and tissue repair, representing a set of plasma proteins that may be used to assess dynamics and extent of

  16. Exhaled Breath Condensate for Proteomic Biomarker Discovery

    Sean W. Harshman

    2014-07-01

    Full Text Available Exhaled breath condensate (EBC has been established as a potential source of respiratory biomarkers. Compared to the numerous small molecules identified, the protein content of EBC has remained relatively unstudied due to the methodological and technical difficulties surrounding EBC analysis. In this review, we discuss the proteins identified in EBC, by mass spectrometry, focusing on the significance of those proteins identified. We will also review the limitations surrounding mass spectral EBC protein analysis emphasizing recommendations to enhance EBC protein identifications by mass spectrometry. Finally, we will provide insight into the future directions of the EBC proteomics field.

  17. Barley seed proteomics from spots to structures

    Finnie, Christine; Svensson, Birte

    2009-01-01

    forms on 2D-gels. Specific protein families, including peroxidases and alpha-amylases have been subjected to in-depth analysis resulting in characterisation of different isozymes, post-translational. modifications and processing. A functional proteomics study focusing on the seed thioredoxin system has...... with information from rice and other cereals facilitate identification of barley proteins. Several hundred barley seed proteins are identified and lower abundance proteins including membrane proteins are now being analysed. In the present review we focus on variation in protein profiles of seed tissues during...

  18. Proteomic approach to nanotoxicity.

    Matysiak, Magdalena; Kapka-Skrzypczak, Lucyna; Brzóska, Kamil; Gutleb, Arno C; Kruszewski, Marcin

    2016-03-30

    In recent years a large number of engineered nanomaterials (NMs) have been developed with promising technical benefits for consumers and medical appliances. In addition to already known potentially advantageous biological properties (antibiotic, antifungal and antiviral activity) of NMs, many new medical applications of NMs are foreseen, such as drug carriers, contrast agents, radiopharmaceuticals and many others. However, there is increasing concern about potential environmental and health effects due to NMs exposure. An increasing body of evidence suggests that NMs may trigger undesirable hazardous interactions with biological systems with potential to generate harmful effects. In this review we summarized a current state of knowledge on the proteomics approaches to nanotoxicity, including protein corona formation, in vitro and in vivo effects of exposure to NMs on proteome of different classes of organisms, from bacteria and plants to mammals. The effects of NMs on the proteome of environmentally relevant organisms are also described. Despite the benefit that development of nanotechnology may bring to the society, there are still major gaps of knowledge on the influence of nanomaterials on human health and the environment. Thus, it seems necessary to conduct further interdisciplinary research to fill the knowledge gaps in NM toxicity, using more holistic approaches than offered by conventional biological techniques. “OMICS” techniques will certainly help researchers in this field. In this paper we summarized the current stage of knowledge of the effects of nanoparticles on the proteome of different organisms, including those commonly used as an environmentally relevant indicator organisms.

  19. Cutting edge proteomics

    Bunkenborg, Jakob; Espadas, Guadalupe; Molina, Henrik

    2013-01-01

    Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC-MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence ...

  20. A proteomic view at T cell costimulation.

    Rudolf Lichtenfels

    Full Text Available The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR through engagement of major histocompatility complex (MHC-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+ CD69(- resting T cells versus cells incubated with (i the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, L-lactate dehydrogenase (LDH, Rho GDP-dissociation inhibitor 2 (GDIR2, and platelet basic protein (CXCL7, were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.

  1. Protein interaction networks by proteome peptide scanning.

    Christiane Landgraf

    2004-01-01

    Full Text Available A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment, that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are

  2. Integrated analysis of transcriptome and proteome changes related to the Ogura cytoplasmic male sterility in cabbage.

    Miaomiao Xing

    Full Text Available Cabbage (Brassica oleracea L. var. capitata, an important vegetable crop in the Brassicaceae family, is economically important worldwide. In the process of hybrid seed production, Ogura cytoplasmic male sterility (OguCMS, controlled by the mitochondrial gene orf138, has been extensively used for cabbage hybrid production with complete and stable male sterility. To identify the critical genes and pathways involved in the sterility and to better understand the underlying molecular mechanisms, the anther of OguCMS line R2P2CMS and the fertile line R2P2 were used for RNA-seq and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation proteome analysis. RNA-seq analysis generated 13,037,109 to 13,066,594 SE50-clean reads, from the sterile and fertile lines, which were assembled into 36,890 unigenes. Among them, 1,323 differentially expressed genes (DEGs were identified, consisting of 307 up- and 1016 down-regulated genes. For ITRAQ analysis, a total of 7,147 unique proteins were identified, and 833 were differentially expressed including 538 up- and 295 down-regulated proteins. These were mainly annotated to the ribosome, spliceosome and mRNA surveillance pathways. Combined transcriptomic and proteomic analyses identified 22 and 70 genes with the same and opposite expression profiles, respectively. Using KEGG analysis of DEGs, gibberellin mediated signaling pathways regulating tapetum programmed cell death and four different pathways involved in sporopollenin synthesis were identified. Secretion and translocation of the sporopollenin precursors were identified, and the key genes participating in these pathways were all significantly down-regulated in R2P2CMS. Light and transmission electron (TE microscopy revealed fat abnormal tapetum rather than vacuolization and degradation at the tetrad and microspore stages of the OguCMS line. This resulted in the failed deposition of sporopollenin on the pollen resulting in sterility. This study provides a

  3. Thermo-msf-parser: an open source Java library to parse and visualize Thermo Proteome Discoverer msf files.

    Colaert, Niklaas; Barsnes, Harald; Vaudel, Marc; Helsens, Kenny; Timmerman, Evy; Sickmann, Albert; Gevaert, Kris; Martens, Lennart

    2011-08-05

    The Thermo Proteome Discoverer program integrates both peptide identification and quantification into a single workflow for peptide-centric proteomics. Furthermore, its close integration with Thermo mass spectrometers has made it increasingly popular in the field. Here, we present a Java library to parse the msf files that constitute the output of Proteome Discoverer. The parser is also implemented as a graphical user interface allowing convenient access to the information found in the msf files, and in Rover, a program to analyze and validate quantitative proteomics information. All code, binaries, and documentation is freely available at http://thermo-msf-parser.googlecode.com.

  4. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

    Ong, S.E.; Blagoev, B.; Kratchmarova, I.

    2002-01-01

    Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. H...

  5. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    Thomas Kieselbach

    Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

  6. Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives.

    Zhou, Li; Wang, Kui; Li, Qifu; Nice, Edouard C; Zhang, Haiyuan; Huang, Canhua

    2016-01-01

    Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy.

  7. The gel electrophoresis markup language (GelML) from the Proteomics Standards Initiative.

    Gibson, Frank; Hoogland, Christine; Martinez-Bartolomé, Salvador; Medina-Aunon, J Alberto; Albar, Juan Pablo; Babnigg, Gyorgy; Wipat, Anil; Hermjakob, Henning; Almeida, Jonas S; Stanislaus, Romesh; Paton, Norman W; Jones, Andrew R

    2010-09-01

    The Human Proteome Organisation's Proteomics Standards Initiative has developed the GelML (gel electrophoresis markup language) data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for MS data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.

  8. iTRAQ-based proteomic analysis reveals alterations in the liver induced by restricted meal frequency in a pig model.

    Liu, Jingbo; Liu, Zhengqun; Chen, Liang; Zhang, Hongfu

    2016-01-01

    The present study was conducted to investigate the effects of meal frequency on metabolite levels in pig plasma and hepatic proteome by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Twenty-four pigs (60.7 ± 1.0 kg) consumed the same amount of feed either in 2 (M2, n = 12) or 12 (M12, n = 12) meals per day. After an 8-wk feeding period, plasma concentrations of metabolites and hormones, hepatic biochemical traits, and proteome (n = 4 per group) were measured. Pigs on the M12 regimen had lower average daily gain and gain-to-feed ratio than pigs fed the M2 regimen. The M2 regimen resulted in lower total lipid, glycogen, and triacylglycerol content in the liver and circulating triacylglycerol concentration than that in the M12 pigs. The metabolic hormone concentrations were not affected by meal frequency, with the exception of elevated fibroblast growth factor 21 concentrations in the M2 regimen compared with the M12 regimen. The iTRAQ-based proteomic analysis revealed 35 differentially expressed proteins in the liver between pigs fed two and 12 meals per day, and these differentially expressed proteins were involved in the regulation of general biological process such as glucose and energy metabolism, lipid metabolism, protein and amino acid metabolism, stress response, and cell redox homeostasis. Altogether, the proteomic results provide insights into the mechanism mediating the beneficial effects of restricted meal frequency on the metabolic fitness. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

    2015-10-30

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  10. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-01-01

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  11. Pilot Study on Mass Spectrometry–Based Analysis of the Proteome of CD34+CD123+ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia

    Johannes R. Schmidt

    2018-02-01

    Full Text Available Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT 10-plex labelling–based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

  12. Mitotic spindle proteomics in Chinese hamster ovary cells.

    Mary Kate Bonner

    Full Text Available Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

  13. iTRAQ-Based Proteomics Analysis of Serum Proteins in Wistar Rats Treated with Sodium Fluoride: Insight into the Potential Mechanism and Candidate Biomarkers of Fluorosis

    Yan Wei

    2016-09-01

    Full Text Available Fluorosis induced by exposure to high level fluoride is quite widespread in the world. The manifestations of fluorosis include dental mottling, bone damage, and impaired malfunction of soft tissues. However, the molecular mechanism of fluorosis has not been clarified until now. To explore the underlying mechanisms of fluorosis and screen out serum biomarkers, we carried out a quantitative proteomics study to identify differentially expressed serum proteins in Wistar rats treated with sodium fluoride (NaF by using a proteomics approach of isobaric tagging for relative and absolute quantitation (iTRAQ. We fed Wistar rats drinking water that had 50, 150, and 250 mg/L of dissolved NaF for 24 weeks. For the experimental duration, each rat was given an examination of the lower incisors to check for the condition of dental fluorosis (DF. By the end of the treatment, fluoride ion concentration in serum and lower incisors were detected. The results showed that NaF treatment can induce rat fluorosis. By iTRAQ analysis, a total of 37 differentially expressed serum proteins were identified between NaF-treated and control rats. These proteins were further analyzed by bioinformatics, out of which two proteins were validated by enzyme-linked immunoadsorbent assays (ELISA. The major proteins were involved in complement and coagulation cascade, inflammatory response, complement activation, defense response, and wound response, suggesting that inflammation and immune reactions may play a key role in fluorosis pathogenesis. These proteins may contribute to the understanding of the mechanism of fluoride toxicity, and may serve as potential biomarkers for fluorosis.

  14. Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits.

    Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin

    2014-06-13

    Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. The potato tuber mitochondrial proteome

    Salvato, Fernanda; Havelund, Jesper Foged; Chen, Mingjie

    2014-01-01

    Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins...... manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome...

  16. Comparative proteomic assessment of matrisome enrichment methodologies

    Krasny, Lukas; Paul, Angela; Wai, Patty; Howard, Beatrice A.; Natrajan, Rachael C.; Huang, Paul H.

    2016-01-01

    The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome. PMID:27589945

  17. Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

    Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

    2016-01-01

    Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

  18. Plant redox proteomics

    Navrot, Nicolas; Finnie, Christine; Svensson, Birte

    2011-01-01

    PTMs in regulating enzymatic activities and controlling biological processes in plants. Notably, proteins controlling the cellular redox state, e.g. thioredoxin and glutaredoxin, appear to play dual roles to maintain oxidative stress resistance and regulate signal transduction pathways via redox PTMs......In common with other aerobic organisms, plants are exposed to reactive oxygen species resulting in formation of post-translational modifications related to protein oxidoreduction (redox PTMs) that may inflict oxidative protein damage. Accumulating evidence also underscores the importance of redox....... To get a comprehensive overview of these types of redox-regulated pathways there is therefore an emerging interest to monitor changes in redox PTMs on a proteome scale. Compared to some other PTMs, e.g. protein phosphorylation, redox PTMs have received less attention in plant proteome analysis, possibly...

  19. PROTEOMICS in aquaculture

    Rodrigues, Pedro M.; Silva, Tomé S.; Dias, Jorge

    2012-01-01

    Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous...... growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance...... questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined...

  20. Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

    Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

    2013-01-01

    Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.

  1. Comparative analysis of methicillin-sensitive and resistant Staphylococcus aureus exposed to emodin based on proteomic profiling.

    Ji, Xiaoyu; Liu, Xiaoqiang; Peng, Yuanxia; Zhan, Ruoting; Xu, Hui; Ge, Xijin

    2017-12-09

    Emodin has a strong antibacterial activity, including methicillin-resistant Staphylococcus aureus (MRSA). However, the mechanism by which emodin induces growth inhibition against MRSA remains unclear. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomics approach was used to investigate the modes of action of emodin on a MRSA isolate and methicillin-sensitive S. aureus ATCC29213(MSSA). Proteomic analysis showed that expression levels of 145 and 122 proteins were changed significantly in MRSA and MSSA, respectively, after emodin treatment. Comparative analysis of the functions of differentially expressed proteins between the two strains was performed via bioinformatics tools blast2go and STRING database. Proteins related to pyruvate pathway imbalance induction, protein synthesis inhibition, and DNA synthesis suppression were found in both methicillin-sensitive and resistant strains. Moreover, Interference proteins related to membrane damage mechanism were also observed in MRSA. Our findings indicate that emodin is a potential antibacterial agent targeting MRSA via multiple mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. The plant mitochondrial proteome

    Millar, A.H.; Heazlewood, J.L.; Kristensen, B.K.

    2005-01-01

    The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid...... context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants....

  3. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Christian Niehage

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

  4. Urine sample preparation for proteomic analysis.

    Olszowy, Pawel; Buszewski, Boguslaw

    2014-10-01

    Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Proteomic characterization of hempseed (Cannabis sativa L.).

    Aiello, Gilda; Fasoli, Elisa; Boschin, Giovanna; Lammi, Carmen; Zanoni, Chiara; Citterio, Attilio; Arnoldi, Anna

    2016-09-16

    This paper presents an investigation on hempseed proteome. The experimental approach, based on combinatorial peptide ligand libraries (CPLLs), SDS-PAGE separation, nLC-ESI-MS/MS identification, and database search, permitted identifying in total 181 expressed proteins. This very large number of identifications was achieved by searching in two databases: Cannabis sativa L. (56 gene products identified) and Arabidopsis thaliana (125 gene products identified). By performing a protein-protein association network analysis using the STRING software, it was possible to build the first interactomic map of all detected proteins, characterized by 137 nodes and 410 interactions. Finally, a Gene Ontology analysis of the identified species permitted to classify their molecular functions: the great majority is involved in the seed metabolic processes (41%), responses to stimulus (8%), and biological process (7%). Hempseed is an underexploited non-legume protein-rich seed. Although its protein is well known for its digestibility, essential amino acid composition, and useful techno-functional properties, a comprehensive proteome characterization is still lacking. The objective of this work was to fill this knowledge gap and provide information useful for a better exploitation of this seed in different food products. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Combining Search Engines for Comparative Proteomics

    Tabb, David

    2012-01-01

    Many proteomics laboratories have found spectral counting to be an ideal way to recognize biomarkers that differentiate cohorts of samples. This approach assumes that proteins that differ in quantity between samples will generate different numbers of identifiable tandem mass spectra. Increasingly, researchers are employing multiple search engines to maximize the identifications generated from data collections. This talk evaluates four strategies to combine information from multiple search engines in comparative proteomics. The “Count Sum” model pools the spectra across search engines. The “Vote Counting” model combines the judgments from each search engine by protein. Two other models employ parametric and non-parametric analyses of protein-specific p-values from different search engines. We evaluated the four strategies in two different data sets. The ABRF iPRG 2009 study generated five LC-MS/MS analyses of “red” E. coli and five analyses of “yellow” E. coli. NCI CPTAC Study 6 generated five concentrations of Sigma UPS1 spiked into a yeast background. All data were identified with X!Tandem, Sequest, MyriMatch, and TagRecon. For both sample types, “Vote Counting” appeared to manage the diverse identification sets most effectively, yielding heightened discrimination as more search engines were added.

  7. Shotgun Proteomics and Biomarker Discovery

    W. Hayes McDonald

    2002-01-01

    Full Text Available Coupling large-scale sequencing projects with the amino acid sequence information that can be gleaned from tandem mass spectrometry (MS/MS has made it much easier to analyze complex mixtures of proteins. The limits of this “shotgun” approach, in which the protein mixture is proteolytically digested before separation, can be further expanded by separating the resulting mixture of peptides prior to MS/MS analysis. Both single dimensional high pressure liquid chromatography (LC and multidimensional LC (LC/LC can be directly interfaced with the mass spectrometer to allow for automated collection of tremendous quantities of data. While there is no single technique that addresses all proteomic challenges, the shotgun approaches, especially LC/LC-MS/MS-based techniques such as MudPIT (multidimensional protein identification technology, show advantages over gel-based techniques in speed, sensitivity, scope of analysis, and dynamic range. Advances in the ability to quantitate differences between samples and to detect for an array of post-translational modifications allow for the discovery of classes of protein biomarkers that were previously unassailable.

  8. Proteomic profiles in hyperandrogenic syndromes.

    Misiti, S; Stigliano, A; Borro, M; Gentile, G; Michienzi, S; Cerquetti, L; Bucci, B; Argese, N; Brunetti, E; Simmaco, M; Toscano, V

    2010-03-01

    Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

  9. Magnetoresistive biosensors for quantitative proteomics

    Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

    2017-08-01

    Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

  10. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Proteomic Markers of Functional Sperm Population in Bovines: Comparison of Low- and High-Density Spermatozoa Following Cryopreservation.

    D'Amours, Olivier; Frenette, Gilles; Bourassa, Sylvie; Calvo, Ézéchiel; Blondin, Patrick; Sullivan, Robert

    2018-01-05

    Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects

  12. Vacuum-assisted breast biopsy of suspected mammographic breast diagnoses: predictive value of serum proteomic profile

    Schittulli, F.; Ventrella, V.

    2009-01-01

    The project planned a series of actions oriented to different scientific questions: to complete the prospective collection of serum samples for serum proteomic analysis according to SOPs needed for the Italy-USA program; the identification of different mammographic signs for prediction of histological diagnosis of breast lesions through mammotone; the analysis of relationship between serum proteomic profile and micro histology characteristics of breast lesions

  13. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

  14. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

  15. Proteomic screening for amyloid proteins.

    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  16. Stressor-induced proteome alterations in zebrafish: A meta-analysis of response patterns

    Groh, Ksenia J., E-mail: ksenia.groh@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, 8093 Zürich (Switzerland); Suter, Marc J.-F. [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Environmental Systems Science, 8092 Zürich (Switzerland)

    2015-02-15

    Highlights: • We compared reported proteome changes induced by various stressors in zebrafish. • Several proteins groups frequently responding to diverse stressors were identified. • These included energy metabolism enzymes, heat shock and cytoskeletal proteins. • Insufficient proteome coverage impedes identification of more specific responses. • Further research needs for proteomics in ecotoxicology are discussed. - Abstract: Proteomics approaches are being increasingly applied in ecotoxicology on the premise that the identification of specific protein expression changes in response to a particular chemical would allow elucidation of the underlying molecular pathways leading to an adverse effect. This in turn is expected to promote the development of focused testing strategies for specific groups of toxicants. Although both gel-based and gel-free global characterization techniques provide limited proteome coverage, the conclusions regarding the cellular processes affected are still being drawn based on the few changes detected. To investigate how specific the detected responses are, we analyzed a set of studies that characterized proteome alterations induced by various physiological, chemical and biological stressors in zebrafish, a popular model organism. Our analysis highlights several proteins and protein groups, including heat shock and oxidative stress defense proteins, energy metabolism enzymes and cytoskeletal proteins, to be most frequently identified as responding to diverse stressors. In contrast, other potentially more specifically responding protein groups are detected much less frequently. Thus, zebrafish proteome responses to stress reported by different studies appear to depend mostly on the level of stress rather than on the specific stressor itself. This suggests that the most broadly used current proteomics technologies do not provide sufficient proteome coverage to allow in-depth investigation of specific mechanisms of toxicant action

  17. Stressor-induced proteome alterations in zebrafish: A meta-analysis of response patterns

    Groh, Ksenia J.; Suter, Marc J.-F.

    2015-01-01

    Highlights: • We compared reported proteome changes induced by various stressors in zebrafish. • Several proteins groups frequently responding to diverse stressors were identified. • These included energy metabolism enzymes, heat shock and cytoskeletal proteins. • Insufficient proteome coverage impedes identification of more specific responses. • Further research needs for proteomics in ecotoxicology are discussed. - Abstract: Proteomics approaches are being increasingly applied in ecotoxicology on the premise that the identification of specific protein expression changes in response to a particular chemical would allow elucidation of the underlying molecular pathways leading to an adverse effect. This in turn is expected to promote the development of focused testing strategies for specific groups of toxicants. Although both gel-based and gel-free global characterization techniques provide limited proteome coverage, the conclusions regarding the cellular processes affected are still being drawn based on the few changes detected. To investigate how specific the detected responses are, we analyzed a set of studies that characterized proteome alterations induced by various physiological, chemical and biological stressors in zebrafish, a popular model organism. Our analysis highlights several proteins and protein groups, including heat shock and oxidative stress defense proteins, energy metabolism enzymes and cytoskeletal proteins, to be most frequently identified as responding to diverse stressors. In contrast, other potentially more specifically responding protein groups are detected much less frequently. Thus, zebrafish proteome responses to stress reported by different studies appear to depend mostly on the level of stress rather than on the specific stressor itself. This suggests that the most broadly used current proteomics technologies do not provide sufficient proteome coverage to allow in-depth investigation of specific mechanisms of toxicant action

  18. Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

    Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; Moore, Ronald J.; Lim, Sujung; Orphan, Victoria J.; Paša-Tolić, Ljiljana; Qian, Wei-Jun; Smith, Richard D.; Kelly, Ryan T.

    2018-04-01

    One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.

  19. Farm animal proteomics - A review

    Bendixen, Emøke; Danielsen, Marianne; Hollung, Kristin

    2011-01-01

    In agricultural sciences as in all other areas of life science, the implementation of proteomics and other post-genomic tools is an important step towards more detailed understanding of the complex biological systems that control physiology and pathology of living beings. Farm animals are raised...... and cattle are relevant not only for farm animal sciences, but also for adding to our understanding of complex biological mechanisms of health and disease in humans. The aim of this review is to present an overview of the specific topics of interest within farm animal proteomics, and to highlight some...... of the areas where synergy between classic model organism proteomics and farm animal proteomics is rapidly emerging. Focus will be on introducing the special biological traits that play an important role in food production, and on how proteomics may help optimize farm animal production...

  20. Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity

    Qian, Haifeng; Lu, Haiping; Ding, Haiyan; Lavoie, Michel; Li, Yali; Liu, Weiping; Fu, Zhengwei

    2015-07-01

    Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.

  1. Quantitative evaluation of the mitochondrial proteomes of Drosophila melanogaster adapted to extreme oxygen conditions.

    Songyue Yin

    Full Text Available Mitochondria are the primary organelles that consume oxygen and provide energy for cellular activities. To investigate the mitochondrial mechanisms underlying adaptation to extreme oxygen conditions, we generated Drosophila strains that could survive in low- or high-oxygen environments (LOF or HOF, respectively, examined their mitochondria at the ultrastructural level via transmission electron microscopy, studied the activity of their respiratory chain complexes, and quantitatively analyzed the protein abundance responses of the mitochondrial proteomes using Isobaric tag for relative and absolute quantitation (iTRAQ. A total of 718 proteins were identified with high confidence, and 55 and 75 mitochondrial proteins displayed significant differences in abundance in LOF and HOF, respectively, compared with the control flies. Importantly, these differentially expressed mitochondrial proteins are primarily involved in respiration, calcium regulation, the oxidative response, and mitochondrial protein translation. A correlation analysis of the changes in the levels of the mRNAs corresponding to differentially regulated mitochondrial proteins revealed two sets of proteins with different modes of regulation (transcriptional vs. post-transcriptional in both LOF and HOF. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to extreme oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.

  2. Proteomic Changes Associated with Successive Reproductive Periods in Male Polychaetous Neanthes arenaceodentata

    Chandramouli, Kondethimmanahalli; Reish, Donald; Zhang, Huoming; Qian, Pei-Yuan; Ravasi, Timothy

    2015-01-01

    The polychaetous annelid Neanthes acuminata complex has a widespread distribution, with the California population referred to as N. arenaceodentata. The reproductive pattern in this complex is unique, in that the female reproduces once and then dies, whereas the male can reproduce up to nine times. The male incubates the embryos until the larvae leave the male’s tube 21–28 days later and commences feeding. Reproductive success and protein expression patterns were measured over the nine reproductive periods. The percent success of the male in producing juveniles increased during the first three reproductive periods and then decreased, but the number of juveniles produced was similar through all nine periods. iTRAQ based quantitative proteomics were used to analyze the dynamics of protein expression patterns. The expression patterns of several proteins were found to be altered. The abundant expression of muscular and contractile proteins may have affected body weight and reproductive success. Sperm have never been observed; fertilization occurs within the parent’s tube. Proteins associated with sperm maturation and fertilization were identified, including ATPase, clathrin, peroxiredoxins and enolase, which may provide clues to the molecular mechanisms enabling males to reproduce multiple times.

  3. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    Yan, Hu; Zhou, Wenhao [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Wei, Liming; Zhong, Fan [Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Roda, Shanghai 200032 (China); Yang, Yi, E-mail: yyang@shmu.edu.cn [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China)

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  4. Proteomic Changes Associated with Successive Reproductive Periods in Male Polychaetous Neanthes arenaceodentata

    Chandramouli, Kondethimmanahalli

    2015-09-04

    The polychaetous annelid Neanthes acuminata complex has a widespread distribution, with the California population referred to as N. arenaceodentata. The reproductive pattern in this complex is unique, in that the female reproduces once and then dies, whereas the male can reproduce up to nine times. The male incubates the embryos until the larvae leave the male’s tube 21–28 days later and commences feeding. Reproductive success and protein expression patterns were measured over the nine reproductive periods. The percent success of the male in producing juveniles increased during the first three reproductive periods and then decreased, but the number of juveniles produced was similar through all nine periods. iTRAQ based quantitative proteomics were used to analyze the dynamics of protein expression patterns. The expression patterns of several proteins were found to be altered. The abundant expression of muscular and contractile proteins may have affected body weight and reproductive success. Sperm have never been observed; fertilization occurs within the parent’s tube. Proteins associated with sperm maturation and fertilization were identified, including ATPase, clathrin, peroxiredoxins and enolase, which may provide clues to the molecular mechanisms enabling males to reproduce multiple times.

  5. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

    2010-01-01

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  6. Expanding the bovine milk proteome through extensive fractionation.

    Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

    2013-01-01

    Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the

  7. Annotating N termini for the human proteome project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome.

    Lange, Philipp F; Huesgen, Pitter F; Nguyen, Karen; Overall, Christopher M

    2014-04-04

    A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as "missing", this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

  8. Proteomics research in India: an update.

    Reddy, Panga Jaipal; Atak, Apurva; Ghantasala, Saicharan; Kumar, Saurabh; Gupta, Shabarni; Prasad, T S Keshava; Zingde, Surekha M; Srivastava, Sanjeeva

    2015-09-08

    After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

    Yang, Xiaoli; Li, Hongtao; Zhang, Chengdong; Lin, Zhidi; Zhang, Xinhua; Zhang, Youjie; Yu, Yanbao; Liu, Kun; Li, Muyan; Zhang, Yuening; Lv, Wenxin; Xie, Yuanliang; Lu, Zheng; Wu, Chunlei; Teng, Ruobing; Lu, Shaoming; He, Min; Mo, Zengnan

    2015-10-01

    Prostatitis is one of the most common urological problems afflicting adult men. The etiology and pathogenesis of nonbacterial prostatitis, which accounts for 90-95% of cases, is largely unknown. As serum proteins often indicate the overall pathologic status of patients, we hypothesized that protein biomarkers of prostatitis might be identified by comparing the serum proteomes of patients with and without nonbacterial prostatitis. All untreated samples were collected from subjects attending the Fangchenggang Area Male Health and Examination Survey (FAMHES). We profiled pooled serum samples from four carefully selected groups of patients (n = 10/group) representing the various categories of nonbacterial prostatitis (IIIa, IIIb, and IV) and matched healthy controls using a mass spectrometry-based 4-plex iTRAQ proteomic approach. More than 160 samples were validated by ELISA. Overall, 69 proteins were identified. Among them, 42, 52, and 37 proteins were identified with differential expression in Category IIIa, IIIb, and IV prostatitis, respectively. The 19 common proteins were related to immunity and defense, ion binding, transport, and proteolysis. Two zinc-binding proteins, superoxide dismutase 3 (SOD3), and carbonic anhydrase I (CA1), were significantly higher in all types of prostatitis than in the control. A receiver operating characteristic curve estimated sensitivities of 50.4 and 68.1% and specificities of 92.1 and 83.8% for CA1 and SOD3, respectively, in detecting nonbacterial prostatitis. The serum CA1 concentration was inversely correlated to the zinc concentration in expressed-prostatic secretions. Our findings suggest that SOD3 and CA1 are potential diagnostic markers of nonbacterial prostatitis, although further large-scale studies are required. The molecular profiles of nonbacterial prostatitis pathogenesis may lay a foundation for discovery of new therapies. © 2015 Wiley Periodicals, Inc.

  10. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Guangqi Li

    Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  11. Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.

    Martínez-Bartolomé, Salvador; Deutsch, Eric W; Binz, Pierre-Alain; Jones, Andrew R; Eisenacher, Martin; Mayer, Gerhard; Campos, Alex; Canals, Francesc; Bech-Serra, Joan-Josep; Carrascal, Montserrat; Gay, Marina; Paradela, Alberto; Navajas, Rosana; Marcilla, Miguel; Hernáez, María Luisa; Gutiérrez-Blázquez, María Dolores; Velarde, Luis Felipe Clemente; Aloria, Kerman; Beaskoetxea, Jabier; Medina-Aunon, J Alberto; Albar, Juan P

    2013-12-16

    Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and

  12. Pollen developmental defects in ZD-CMS rice line explored by cytological, molecular and proteomic approaches.

    Yan, Junjie; Tian, Han; Wang, Shuzhen; Shao, Jinzhen; Zheng, Yinzhen; Zhang, Hongyuan; Guo, Lin; Ding, Yi

    2014-08-28

    Cytoplasmic male sterility (CMS) is a widely observed phenomenon, which is especially useful in hybrid seed production. Meixiang A (MxA) is a new rice CMS line derived from a pollen-free sterile line named Yunnan ZidaoA (ZD-CMS). In this study, a homologous WA352 gene with variation in two nucleotides was identified in MxA. Cytological analysis revealed that MxA was aborted in the early uninucleate stage. The protein expression profiles of MxA and its maintainer line MeixiangB (MxB) were systematically compared using iTRAQ-based quantitative proteomics technology using young florets at the early uninucleate stage. A total of 688 proteins were quantified in both rice lines, and 45 of these proteins were found to be differentially expressed. Bioinformatics analysis indicated a large number of the proteins involved in carbohydrate metabolism or the stress response were downregulated in MxA, suggesting that these metabolic processes had been hindered during pollen development in MxA. The ROS (reactive oxygen species) level was increased in the mitochondrion of MxA, and further ultrastructural analysis showed the mitochondria with disrupted cristae in the rice CMS line MxA. These findings substantially contribute to our knowledge of pollen developmental defects in ZD-CMS rice line. MeixiangA (MxA) is a new type of rice CMS line, which is derived from pollen-free sterile line Yunnan ZidaoA. In this study, the cytological, molecular and proteomic approaches were used to study the characteristics of this new CMS line. Cytological study indicates the CMS line is aborted at the early uninucleate stage. A potential sterile gene ZD352 is identified in MxA, the protein product of which is mainly accumulated at the MMC/Meiotic stage. iTRAQ based proteomic analysis is performed to study the relevant proteins involved in the CMS occurance, 45 proteins are found to be significant differentially expressed and these proteins are involved in many cellular processes such as

  13. Proteomics of Maize Root Development.

    Hochholdinger, Frank; Marcon, Caroline; Baldauf, Jutta A; Yu, Peng; Frey, Felix P

    2018-01-01

    Maize forms a complex root system with structurally and functionally diverse root types that are formed at different developmental stages to extract water and mineral nutrients from soil. In recent years proteomics has been intensively applied to identify proteins involved in shaping the three-dimensional architecture and regulating the function of the maize root system. With the help of developmental mutants, proteomic changes during the initiation and emergence of shoot-borne, lateral and seminal roots have been examined. Furthermore, root hairs were surveyed to understand the proteomic changes during the elongation of these single cell type structures. In addition, primary roots have been used to study developmental changes of the proteome but also to investigate the proteomes of distinct tissues such as the meristematic zone, the elongation zone as well as stele and cortex of the differentiation zone. Moreover, subcellular fractions of the primary root including cell walls, plasma membranes and secreted mucilage have been analyzed. Finally, the superior vigor of hybrid seedling roots compared to their parental inbred lines was studied on the proteome level. In summary, these studies provide novel insights into the complex proteomic interactions of the elaborate maize root system during development.

  14. Proteomics of Maize Root Development

    Frank Hochholdinger

    2018-03-01

    Full Text Available Maize forms a complex root system with structurally and functionally diverse root types that are formed at different developmental stages to extract water and mineral nutrients from soil. In recent years proteomics has been intensively applied to identify proteins involved in shaping the three-dimensional architecture and regulating the function of the maize root system. With the help of developmental mutants, proteomic changes during the initiation and emergence of shoot-borne, lateral and seminal roots have been examined. Furthermore, root hairs were surveyed to understand the proteomic changes during the elongation of these single cell type structures. In addition, primary roots have been used to study developmental changes of the proteome but also to investigate the proteomes of distinct tissues such as the meristematic zone, the elongation zone as well as stele and cortex of the differentiation zone. Moreover, subcellular fractions of the primary root including cell walls, plasma membranes and secreted mucilage have been analyzed. Finally, the superior vigor of hybrid seedling roots compared to their parental inbred lines was studied on the proteome level. In summary, these studies provide novel insights into the complex proteomic interactions of the elaborate maize root system during development.

  15. Proteomic Signatures of Thymomas.

    Linan Wang

    Full Text Available Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas.

  16. Proteomic Analysis Reveals Coordinated Regulation of Anthocyanin Biosynthesis through Signal Transduction and Sugar Metabolism in Black Rice Leaf.

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zheng, Jingui

    2017-12-15

    Black rice ( Oryza sativa L.) is considered to be a healthy food due to its high content of anthocyanins in the pericarp. The synthetic pathway of anthocyanins in black rice grains has been identified, however, the proteomic profile of leaves during grain development is still unclear. Here, isobaric Tags Relative and Absolute Quantification (iTRAQ) MS/MS was carried out to identify statistically significant changes of leaf proteome in the black rice during grain development. Throughout three sequential developmental stages, a total of 3562 proteins were detected and 24 functional proteins were differentially expressed 3-10 days after flowering (DAF). The detected proteins are known to be involved in various biological processes and most of these proteins were related to gene expression regulatory (33.3%), signal transduction (16.7%) and developmental regulation and hormone-like proteins (12.5%). The coordinated changes were consistent with changes in regulatory proteins playing a leading role in leaves during black rice grain development. This indicated that signal transduction between leaves and grains may have an important role in anthocyanin biosynthesis and accumulation during grain development of black rice. In addition, four identified up-regulated proteins associated with starch metabolism suggested that the remobilization of nutrients for starch synthesis plays a potential role in anthocyanin biosynthesis of grain. The mRNA transcription for eight selected proteins was validated with quantitative real-time PCR. Our results explored the proteomics of the coordination between leaf and grain in anthocyanins biosynthesis of grain, which might be regulated by signal transduction and sugar metabolism in black rice leaf.

  17. Proteomic Analysis Reveals Coordinated Regulation of Anthocyanin Biosynthesis through Signal Transduction and Sugar Metabolism in Black Rice Leaf

    Linghua Chen

    2017-12-01

    Full Text Available Black rice (Oryza sativa L. is considered to be a healthy food due to its high content of anthocyanins in the pericarp. The synthetic pathway of anthocyanins in black rice grains has been identified, however, the proteomic profile of leaves during grain development is still unclear. Here, isobaric Tags Relative and Absolute Quantification (iTRAQ MS/MS was carried out to identify statistically significant changes of leaf proteome in the black rice during grain development. Throughout three sequential developmental stages, a total of 3562 proteins were detected and 24 functional proteins were differentially expressed 3–10 days after flowering (DAF. The detected proteins are known to be involved in various biological processes and most of these proteins were related to gene expression regulatory (33.3%, signal transduction (16.7% and developmental regulation and hormone-like proteins (12.5%. The coordinated changes were consistent with changes in regulatory proteins playing a leading role in leaves during black rice grain development. This indicated that signal transduction between leaves and grains may have an important role in anthocyanin biosynthesis and accumulation during grain development of black rice. In addition, four identified up-regulated proteins associated with starch metabolism suggested that the remobilization of nutrients for starch synthesis plays a potential role in anthocyanin biosynthesis of grain. The mRNA transcription for eight selected proteins was validated with quantitative real-time PCR. Our results explored the proteomics of the coordination between leaf and grain in anthocyanins biosynthesis of grain, which might be regulated by signal transduction and sugar metabolism in black rice leaf.

  18. Comparison of serum fractionation methods by data independent label-free proteomics

    D. Baiwir

    2015-12-01

    Full Text Available Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values.

  19. Characterization of the porcine synovial fluid proteome and a comparison to the plasma proteome

    Tue Bjerg Bennike

    2015-12-01

    In addition, we analyzed the proteome of human plasma, and compared the proteomes to the obtained porcine synovial fluid proteome. The proteome of the two body fluids were found highly similar, underlining the detected plasma derived nature of many synovial fluid components. The healthy porcine synovial fluid proteomics data, human rheumatoid arthritis synovial fluid proteomics data used in the method optimization, human plasma proteomics data, and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD000935.

  20. The Proteomics Big Challenge for Biomarkers and New Drug-Targets Discovery

    Savino, Rocco; Paduano, Sergio; Preianò, Mariaimmacolata; Terracciano, Rosa

    2012-01-01

    In the modern process of drug discovery, clinical, functional and chemical proteomics can converge and integrate synergies. Functional proteomics explores and elucidates the components of pathways and their interactions which, when deregulated, lead to a disease condition. This knowledge allows the design of strategies to target multiple pathways with combinations of pathway-specific drugs, which might increase chances of success and reduce the occurrence of drug resistance. Chemical proteomics, by analyzing the drug interactome, strongly contributes to accelerate the process of new druggable targets discovery. In the research area of clinical proteomics, proteome and peptidome mass spectrometry-profiling of human bodily fluid (plasma, serum, urine and so on), as well as of tissue and of cells, represents a promising tool for novel biomarker and eventually new druggable targets discovery. In the present review we provide a survey of current strategies of functional, chemical and clinical proteomics. Major issues will be presented for proteomic technologies used for the discovery of biomarkers for early disease diagnosis and identification of new drug targets. PMID:23203042

  1. Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

    Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

    2013-05-01

    Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

  2. Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

    Sorette M

    2004-12-01

    Full Text Available Abstract Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

  3. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

    Zhang, Yanling; Zhang, Yong; Adachi, Jun

    2007-01-01

    and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http......://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic...... annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools....

  4. Evaluation of sample extraction methods for proteomics analysis of green algae Chlorella vulgaris.

    Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

    2016-05-01

    Many protein extraction methods have been developed for plant proteome analysis but information is limited on the optimal protein extraction method from algae species. This study evaluated four protein extraction methods, i.e. direct lysis buffer method, TCA-acetone method, phenol method, and phenol/TCA-acetone method, using green algae Chlorella vulgaris for proteome analysis. The data presented showed that phenol/TCA-acetone method was superior to the other three tested methods with regards to shotgun proteomics. Proteins identified using shotgun proteomics were validated using sequential window acquisition of all theoretical fragment-ion spectra (SWATH) technique. Additionally, SWATH provides protein quantitation information from different methods and protein abundance using different protein extraction methods was evaluated. These results highlight the importance of green algae protein extraction method for subsequent MS analysis and identification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Plant plasma membrane proteomics for improving cold tolerance

    Daisuke eTakahashi

    2013-04-01

    Full Text Available Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation. One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

  6. The hemolymph proteome of fed and starved Drosophila larvae.

    Handke, Björn; Poernbacher, Ingrid; Goetze, Sandra; Ahrens, Christian H; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F

    2013-01-01

    The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

  7. The hemolymph proteome of fed and starved Drosophila larvae.

    Björn Handke

    Full Text Available The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

  8. Proteomics data repositories: Providing a safe haven for your data and acting as a springboard for further research

    Vizcaíno, Juan Antonio; Foster, Joseph M.; Martens, Lennart

    2010-01-01

    Despite the fact that data deposition is not a generalised fact yet in the field of proteomics, several mass spectrometry (MS) based proteomics repositories are publicly available for the scientific community. The main existing resources are: the Global Proteome Machine Database (GPMDB), PeptideAtlas, the PRoteomics IDEntifications database (PRIDE), Tranche, and NCBI Peptidome. In this review the capabilities of each of these will be described, paying special attention to four key properties: data types stored, applicable data submission strategies, supported formats, and available data mining and visualization tools. Additionally, the data contents from model organisms will be enumerated for each resource. There are other valuable smaller and/or more specialized repositories but they will not be covered in this review. Finally, the concept behind the ProteomeXchange consortium, a collaborative effort among the main resources in the field, will be introduced. PMID:20615486

  9. Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

    Lo, Andy; Weiner, Joel H.; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

  10. Proteomics of Skeletal Muscle

    Deshmukh, Atul

    2016-01-01

    , of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle......Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability...... of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence...

  11. The Succinated Proteome

    Merkley, Eric D.; Metz, Thomas O.; Smith, Richard D.; Baynes, John; Frizell, Norma

    2014-03-30

    Succination is a chemical modification of cysteine in protein by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes. Increased succination of proteins is also detected in the kidney of a fumarase conditional knock-out mouse which develops renal tumors. Keap1, the gatekeeper of the antioxidant response, was identified as a major succinated protein in renal cancer cells, suggesting that succination may play a role in activation of the antioxidant response. A wide range of proteins is subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones with functional cysteine residues. There is also significant overlap between succinated and glutathionylated proteins, and with proteins containing cysteine residues that are readily oxidized to the sulfenic (cysteic) acid. Succination of adipocyte proteins is inhibited by uncouplers, which discharge the mitochondrial membrane potential (Δψm) and by ER stress inhibitors. 2SC serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non