High throughput and accurate serum proteome profiling by integrated sample preparation technology and single-run data independent mass spectrometry analysis.

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Lin, Lin; Zheng, Jiaxin; Yu, Quan; Chen, Wendong; Xing, Jinchun; Chen, Chenxi; Tian, Ruijun

2018-03-01

Mass spectrometry (MS)-based serum proteome analysis is extremely challenging due to its high complexity and dynamic range of protein abundances. Developing high throughput and accurate serum proteomic profiling approach capable of analyzing large cohorts is urgently needed for biomarker discovery. Herein, we report a streamlined workflow for fast and accurate proteomic profiling from 1μL of blood serum. The workflow combined an integrated technique for highly sensitive and reproducible sample preparation and a new data-independent acquisition (DIA)-based MS method. Comparing with standard data dependent acquisition (DDA) approach, the optimized DIA method doubled the number of detected peptides and proteins with better reproducibility. Without protein immunodepletion and prefractionation, the single-run DIA analysis enables quantitative profiling of over 300 proteins with 50min gradient time. The quantified proteins span more than five orders of magnitude of abundance range and contain over 50 FDA-approved disease markers. The workflow allowed us to analyze 20 serum samples per day, with about 358 protein groups per sample being identified. A proof-of-concept study on renal cell carcinoma (RCC) serum samples confirmed the feasibility of the workflow for large scale serum proteomic profiling and disease-related biomarker discovery. Blood serum or plasma is the predominant specimen for clinical proteomic studies while the analysis is extremely challenging for its high complexity. Many efforts had been made in the past for serum proteomics for maximizing protein identifications, whereas few have been concerned with throughput and reproducibility. Here, we establish a rapid, robust and high reproducible DIA-based workflow for streamlined serum proteomic profiling from 1μL serum. The workflow doesn't need protein depletion and pre-fractionation, while still being able to detect disease-relevant proteins accurately. The workflow is promising in clinical application

A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

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Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

2012-12-21

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones.

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El Kennani, Sara; Adrait, Annie; Shaytan, Alexey K; Khochbin, Saadi; Bruley, Christophe; Panchenko, Anna R; Landsman, David; Pflieger, Delphine; Govin, Jérôme

2017-01-01

Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the "HistoneDB2.0 with Variants" database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants.

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

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Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

2017-09-01

The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for

Bioinformatical Analysis of Organ-Related (Heart, Brain, Liver, and Kidney and Serum Proteomic Data to Identify Protein Regulation Patterns and Potential Sepsis Biomarkers

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Andreas Hohn

2018-01-01

Full Text Available During the last years, proteomic studies have revealed several interesting findings in experimental sepsis models and septic patients. However, most studies investigated protein alterations only in single organs or in whole blood. To identify possible sepsis biomarkers and to evaluate the relationship between protein alteration in sepsis affected organs and blood, proteomics data from the heart, brain, liver, kidney, and serum were analysed. Using functional network analyses in combination with hierarchical cluster analysis, we found that protein regulation patterns in organ tissues as well as in serum are highly dynamic. In the tissue proteome, the main functions and pathways affected were the oxidoreductive activity, cell energy generation, or metabolism, whereas in the serum proteome, functions were associated with lipoproteins metabolism and, to a minor extent, with coagulation, inflammatory response, and organ regeneration. Proteins from network analyses of organ tissue did not correlate with statistically significantly regulated serum proteins or with predicted proteins of serum functions. In this study, the combination of proteomic network analyses with cluster analyses is introduced as an approach to deal with high-throughput proteomics data to evaluate the dynamics of protein regulation during sepsis.

Investigation of heart proteome of different consomic mouse strains. Testing the effect of polymorphisms on the proteome-wide trans-variation of proteins

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Stefanie Forler

2015-06-01

Full Text Available We investigated to which extent polymorphisms of an individual affect the proteomic network. Consomic mouse strains (CS were used to study the trans-effect of the cis-variant (polymorphic proteins of the strain PWD/Ph on the proteins of the host strain C57BL/6J. The cardiac proteome of ten CSs was analyzed by 2-DE and MS. Cis-variant PWD proteins altered a high number of C57BL/6J proteins, but the number of trans-variant proteins differed considerably between different CSs. Cardiac hypertrophy was induced in CSs. We found that high variability of the proteome, as induced by polymorphisms in CS14, acts protective against the complex disease.

Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics

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Nie Jing

2011-05-01

Full Text Available Abstract Background High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. Results The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. Conclusions The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.

Relationship between radiobiological hypoxia in a C3H mouse mammary carcinoma and osteopontin levels in mouse serum

DEFF Research Database (Denmark)

Lukácová, Slávka; Khalil, Azza Ahmed; Overgaard, Jens

2005-01-01

To investigate the possible relationship between radiobiological hypoxia in a C3H mouse mammary carcinoma and osteopontin (OPN) levels measured in mouse serum. MATERIAL AND METHODS: Experiments were performed in CDF1 mice that were either non-tumour bearing or with different sized tumours implanted...... in the right rear foot. Osteopontin levels in extracted mouse blood serum and tissue from the transplanted tumours were measured using an ELISA assay. The tumour oxygenation status was estimated using the Eppendorf Histograph and the fraction of oxygen partial pressure (pO2) values =5 mm Hg (HF5...

Analysis of proteomic changes of the serum of irradiated mice

International Nuclear Information System (INIS)

Wang Zhidong; Chen Xiaohua; Dong Bo; Zhang Junquan; Rao Yalan; Gao Ronglian; Hou Lili; Mao Bingzhi

2005-01-01

To explore the early diagnostic factors, new therapeutic targets and mechanisms of acute radiation disease. Proteomic changes of the serum of irradiated mice were studied using 2-DE and Q-TOF-MS approaches. One higher level expressed protein after the irradiation was found, and it was identified as α chain of haptoglobin by Q-TOF-MS. The authors confirmed the result by Western blotting with anti-haptoglobin antibody. Haptoglobin may involve in the process of acute radiation injury. (authors)

Proteomic interactions in the mouse vitreous-retina complex.

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Jessica M Skeie

Full Text Available Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.

Proteomic interactions in the mouse vitreous-retina complex.

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Skeie, Jessica M; Mahajan, Vinit B

2013-01-01

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina. Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software. We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor. Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.

[The proteomic profiling of blood serum of children with gastroesophageal reflux disease].

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Korkotashvili, L V; Kolesov, S A; Jukova, E A; Vidmanova, T A; Kankova, N Yu; Bashurova, I A; Sidorova, A M; Kulakova, E V

2015-03-01

The mass-spectra of proteome of blood serum from healthy children and children with gastroesophageal reflux disease were received. The technology platform including direct proteome mass-spectrometer profiling after pre-fractional rectification using magnetic particles MB WCX was applied. The significant differences in mass-spectra were established manifesting in detection of more mass-spectrometer peaks and higher indicators of their intensity and area in group of healthy children. The study detected 39 particular peptides and low-molecular proteins predominantly intrinsic to healthy or ill children. It was established that two peptides with molecular mass 925 and 909 Da. are registered only in healthy patients and have no traces in group ofpatients with gastroesophageal reflux disease. The peptide 1564 Da is detected only in blood of children with gastroesophageal reflux disease and totally is absent in healthy children. The research data permitted to reveal specific patterns (signatures) of low-molecular proteins and peptides specific for blood serum of healthy children and patients with gastroesophageal reflux disease. The results testify the availability of singularities in metabolism of low-molecular proteins and can be used as a basis for development of minimally invasive mass-spectrometer system for its diagnostic.

Proteomics Analysis for Finding Serum Markers of Ovarian Cancer

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Yushan Cheng

2014-01-01

Full Text Available A combination of peptide ligand library beads (PLLB and 1D gel liquid chromatography-mass spectrometry/mass spectrometry (1DGel-LC-MS/MS was employed to analyze serum samples from patients with ovarian cancer and from healthy controls. Proteomic analysis identified 1200 serum proteins, among which 57 proteins were upregulated and 10 were downregulated in the sera from cancer patients. Retinol binding protein 4 (RBP4 is highly upregulated in the ovarian cancer serum samples. ELISA was employed to measure plasma concentrations of RBP4 in 80 samples from ovarian cancer patients, healthy individuals, myoma patients, and patients with benign ovarian tumor, respectively. The plasma concentrations of RBP4 ranging from 76.91 to 120.08 ng/mL with the mean value 89.13±1.67 ng/mL in ovarian cancer patients are significantly higher than those in healthy individuals (10.85±2.38 ng/mL. Results were further confirmed with immunohistochemistry, demonstrating that RBP4 expression levels in normal ovarian tissue were lower than those in ovarian cancer tissues. Our results suggested that RBP4 is a potential biomarker for diagnostic of screening ovarian cancer.

Diagnosis of major depressive disorder by combining multimodal information from heart rate dynamics and serum proteomics using machine-learning algorithm.

Science.gov (United States)

Kim, Eun Young; Lee, Min Young; Kim, Se Hyun; Ha, Kyooseob; Kim, Kwang Pyo; Ahn, Yong Min

2017-06-02

Major depressive disorder (MDD) is a systemic and multifactorial disorder that involves abnormalities in multiple biochemical pathways and the autonomic nervous system. This study applied a machine-learning method to classify MDD and control groups by incorporating data from serum proteomic analysis and heart rate variability (HRV) analysis for the identification of novel peripheral biomarkers. The study subjects consisted of 25 drug-free female MDD patients and 25 age- and sex-matched healthy controls. First, quantitative serum proteome profiles were analyzed by liquid chromatography-tandem mass spectrometry using pooled serum samples from 10 patients and 10 controls. Next, candidate proteins were quantified with multiple reaction monitoring (MRM) in 50 subjects. We also analyzed 22 linear and nonlinear HRV parameters in 50 subjects. Finally, we identified a combined biomarker panel consisting of proteins and HRV indexes using a support vector machine with recursive feature elimination. A separation between MDD and control groups was achieved using five parameters (apolipoprotein B, group-specific component, ceruloplasmin, RMSSD, and SampEn) at 80.1% classification accuracy. A combination of HRV and proteomic data achieved better classification accuracy. A high classification accuracy can be achieved by combining multimodal information from heart rate dynamics and serum proteomics in MDD. Our approach can be helpful for accurate clinical diagnosis of MDD. Further studies using larger, independent cohorts are needed to verify the role of these candidate biomarkers for MDD diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

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Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Molecular analysis of serum and bronchoalveolar lavage in a mouse model of influenza reveals markers of disease severity that can be clinically useful in humans.

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Yadunanda Kumar

Full Text Available BACKGROUND: Management of influenza, a major contributor to the worldwide disease burden, is complicated by lack of reliable methods for early identification of susceptible individuals. Identification of molecular markers that can augment existing diagnostic tools for prediction of severity can be expected to greatly improve disease management capabilities. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed cytokines, proteome flux and protein adducts in bronchoalveolar lavage (BAL and sera from mice infected with influenza A virus (PR8 strain using a previously established non-lethal model of influenza infection. Through detailed cytokine and protein adduct measurements of murine BAL, we first established the temporal profile of innate and adaptive responses as well as macrophage and neutrophil activities in response to influenza infection. A similar analysis was also performed with sera from a longitudinal cohort of influenza patients. We then used an iTRAQ-based, comparative serum proteome analysis to catalog the proteome flux in the murine BAL during the stages correlating with "peak viremia," "inflammatory damage," as well as the "recovery phase." In addition to activation of acute phase responses, a distinct class of lung proteins including surfactant proteins was found to be depleted from the BAL coincident with their "appearance" in the serum, presumably due to leakage of the protein following loss of the integrity of the lung/epithelial barrier. Serum levels of at least two of these proteins were elevated in influenza patients during the febrile phase of infection compared to healthy controls or to the same patients at convalescence. CONCLUSIONS/SIGNIFICANCE: The findings from this study provide a molecular description of disease progression in a mouse model of influenza and demonstrate its potential for translation into a novel class of markers for measurement of acute lung injury and improved case management.

Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

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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.

Characterization of potential ionizing radiation biomarkers by a proteomic approach

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Guipaud, O; Vereycken-Holler, V; Benderitter, M [Institut de Radioprotection et de Surete Nucleaire, Lab. de Radiopathologie, 92 - Fontenay aux Roses (France); Royer, N; Vinh, J [Ecole Superieure de Physique et de Chimie Industrielles, 75 - Paris (France)

2006-07-01

Radio-induced lesions are tissue specific, hardly predictable, and can arise months or years later. The finding of prognostic bio-markers is of fundamental relevance for the settlement of therapeutic or preventive strategies. Using two-dimensional gel electrophoresis and mass spectrometry, a proteomic study was applied to look for differentially expressed proteins, i.e. potential bio-markers candidates, in mouse serums after a local irradiation of the dorsal skin. Our results clearly indicated that serum protein content was dynamically modified after a local skin irradiation. A set of specific proteins were early down- or up-regulated and could turn out to be good candidates as diagnostic or prognostic bio-markers. (author)

Characterization of potential ionizing radiation biomarkers by a proteomic approach

International Nuclear Information System (INIS)

Guipaud, O.; Vereycken-Holler, V.; Benderitter, M.; Royer, N.; Vinh, J.

2006-01-01

Radio-induced lesions are tissue specific, hardly predictable, and can arise months or years later. The finding of prognostic bio-markers is of fundamental relevance for the settlement of therapeutic or preventive strategies. Using two-dimensional gel electrophoresis and mass spectrometry, a proteomic study was applied to look for differentially expressed proteins, i.e. potential bio-markers candidates, in mouse serums after a local irradiation of the dorsal skin. Our results clearly indicated that serum protein content was dynamically modified after a local skin irradiation. A set of specific proteins were early down- or up-regulated and could turn out to be good candidates as diagnostic or prognostic bio-markers. (author)

Proteomic analysis of mouse thymoma EL4 cells treated with bis (tri-n-butyltin)oxide (TBTO)

NARCIS (Netherlands)

Osman, A.M.; Kol, S.; Peijnenburg, A.A.C.M.; Blokland, M.H.; Pennings, J.L.A.; Kleinjans, J.C.S.; Loveren, van H.

2009-01-01

Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the

Tributyltin Exposure Alters Cytokine Levels in Mouse Serum

Science.gov (United States)

Lawrence, Shanieek; Pellom, Samuel T.; Shanker, Anil; Whalen, Margaret M.

2016-01-01

Tributyltin (TBT), a toxic environmental contaminant, has been widely utilized for various industrial, agricultural and household purposes. Its usage has led to a global contamination and its bioaccumulation in aquatic organisms and terrestrial mammals. Previous studies suggest that TBT has debilitating effects on the overall immune function of animals, rendering them more vulnerable to diseases. TBT (at concentrations that have been detected in human blood) alters secretion of inflammatory cytokines from human lymphocytes ex vivo. Thus, it is important to determine if specified levels of TBT can alter levels of cytokines in an in vivo system. Mice were exposed to biologically relevant concentrations of TBT (200, 100 or 25 nM final concentrations). The quantitative determination of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL2, IL5, IL7, IL12βp40, IL13, IL15, KC, MIP1β, MIP2 and RANTES was performed in mouse sera by MAGPIX analysis and Western blot. Results indicated alterations (both decreases and increases) in several cytokines. The pro-inflammatory cytokines IFNγ, TNFα, IL-1β, IL-2, IL5, IL12βp40, and IL-15 were altered as were the chemokines MIP-1 and RANTES and the anti-inflammatory cytokine IL-13. Increases in IFNγ and TNFα were seen in serum of mice exposed to TBT for less than 24 hr. IL1-β, IL-12βp40, IL-5 and IL-15 were also modulated in mouse serum depending on the specific experiment and the exposure concentration. IL-2 was consistently decreased in mouse serum when animals were exposed to TBT. There were also TBT-induced increases in MIP-1β, RANTES, and IL-13. These results from human and murine samples clearly suggest that TBT exposures modulate the secretion inflammatory cytokines. PMID:27602597

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

Science.gov (United States)

Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

2015-01-01

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

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Jae Eun Lee

2015-06-01

Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

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

Science.gov (United States)

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.

Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

Science.gov (United States)

Borth, N; Heider, R; Assadian, A; Katinger, H

1992-09-01

The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

Science.gov (United States)

Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

Science.gov (United States)

Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

2011-10-01

Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

International Nuclear Information System (INIS)

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

Energy Technology Data Exchange (ETDEWEB)

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.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

International Nuclear Information System (INIS)

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.

Serum Is Not Necessary for Prior Pharmacological Activation of AMPK to Increase Insulin Sensitivity of Mouse Skeletal Muscle

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Nicolas O. Jørgensen

2018-04-01

Full Text Available Exercise, contraction, and pharmacological activation of AMP-activated protein kinase (AMPK by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR have all been shown to increase muscle insulin sensitivity for glucose uptake. Intriguingly, improvements in insulin sensitivity following contraction of isolated rat and mouse skeletal muscle and prior AICAR stimulation of isolated rat skeletal muscle seem to depend on an unknown factor present in serum. One study recently questioned this requirement of a serum factor by showing serum-independency with muscle from old rats. Whether a serum factor is necessary for prior AICAR stimulation to increase insulin sensitivity of mouse skeletal muscle is not known. Therefore, we investigated the necessity of serum for this effect of AICAR in mouse skeletal muscle. We found that the ability of prior AICAR stimulation to improve insulin sensitivity of mouse skeletal muscle did not depend on the presence of serum during AICAR stimulation. Although prior AICAR stimulation did not enhance proximal insulin signaling, insulin-stimulated phosphorylation of Tre-2/BUB2/CDC16- domain family member 4 (TBC1D4 Ser711 was greater in prior AICAR-stimulated muscle compared to all other groups. These results imply that the presence of a serum factor is not necessary for prior AMPK activation by AICAR to enhance insulin sensitivity of mouse skeletal muscle.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

Science.gov (United States)

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.

Tributyltin exposure alters cytokine levels in mouse serum.

Science.gov (United States)

Lawrence, Shanieek; Pellom, Samuel T; Shanker, Anil; Whalen, Margaret M

2016-11-01

Tributyltin (TBT), a toxic environmental contaminant, has been widely utilized for various industrial, agricultural and household purposes. Its usage has led to a global contamination and its bioaccumulation in aquatic organisms and terrestrial mammals. Previous studies suggest that TBT has debilitating effects on the overall immune function of animals, rendering them more vulnerable to diseases. TBT (at concentrations that have been detected in human blood) alters secretion of inflammatory cytokines from human lymphocytes ex vivo. Thus, it is important to determine if specified levels of TBT can alter levels of cytokines in an in vivo system. Mice were exposed to biologically relevant concentrations of TBT (200, 100 or 25 nM final concentrations). The quantitative determination of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL2, IL5, IL7, IL12βp40, IL13, IL15, keratinocyte chemoattractant (KC), macrophage inflammatory protein 1β (MIP), MIP2 and regulated on activation normal T-cell-expressed and secreted (RANTES) was performed in mouse sera by MAGPIX analysis and Western blot. Results indicated alterations (both decreases and increases) in several cytokines. The pro-inflammatory cytokines IFNγ, TNFα, IL-1β, IL-2, IL5, IL12βp40 and IL-15 were altered as were the chemokines MIP-1 and RANTES and the anti-inflammatory cytokine IL-13. Increases in IFNγ and TNFα were seen in the serum of mice exposed to TBT for less than 24 h. Levels of IL1β, IL-12 βp40, IL-5 and IL-15 were also modulated in mouse serum, depending on the specific experiment and exposure level. IL-2 was consistently decreased in mouse serum when animals were exposed to TBT. There were also TBT-induced increases in MIP-1β, RANTES and IL-13. These results from human and murine samples clearly suggest that TBT exposures modulate the secretion inflammatory cytokines.

Distinctive serum miRNA profile in mouse models of striated muscular pathologies.

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Nicolas Vignier

Full Text Available Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD, limb-girdle muscular dystrophy type 2D (LGMD2D, limb-girdle muscular dystrophy type 2C (LGMD2C, Emery-Dreifuss muscular dystrophy (EDMD and hypertrophic cardiomyopathy (HCM. Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.

Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

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Yansheng Liu

Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Science.gov (United States)

Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

2018-01-01

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes

Energy Technology Data Exchange (ETDEWEB)

Zhang, Qibin; Fillmore, Thomas L.; Schepmoes, Athena A.; Clauss, Therese RW; Gritsenko, Marina A.; Mueller, Patricia W.; Rewers, Marian; Atkinson, Mark A.; Smith, Richard D.; Metz, Thomas O.

2013-01-14

Using global liquid chromatography-mass spectrometry (LC-MS)-based proteomics analyses, we identified 24 serum proteins significantly variant between those with type 1 diabetes and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects, and 16 peptides were verified having very good discriminating power, with areas under the receiver operator characteristic curve ≥ 0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetic) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using serum from 50 age matched type 2 diabetic individuals, and a subset of proteins, particularly C1 inhibitor were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with type 1 diabetes from healthy control and type 2 diabetes suggests dysregulated innate immune responses may be associated with the development of this disorder.

A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics

DEFF Research Database (Denmark)

Eberl, H.C.; Mann, M.; Spruijt, C.G.

2013-01-01

Posttranslational modifications on core histones can serve as binding scaffolds for chromatin-associated proteins. Proteins that specifically bind to or "read" these modifications were previously identified in mass spectrometry-based proteomics screens based on stable isotope-labeling in cell lines...... the chromatin interaction landscape in mouse tissues, our workflow can be used for peptides with different modifications and cell types of any organism....

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

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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.

Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series

NARCIS (Netherlands)

Roelofsen, Johan; Alvarez Llamas, Gloria; Dijkstra, Martijn; Breitling, Rainer; Havenga, Klaas; Bijzet, Johannes; Zandbergen, Wouter; de Vries, Marcel; Ploeg, Rutger J.; Vonk, Roel J.

Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.Roelofsen H, Alvarez-Llamas G, Dijkstra M, Breitling R, Havenga K, Bijzet J, Zandbergen W, de Vries MP, Ploeg RJ, Vonk RJ. Centre for Medical Biomics, University Medical Centre

Effect of Rat Medicated Serum Containing You Gui Wan on Mouse Oocyte In Vitro Maturation and Subsequent Fertilization Competence

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Xiao-Hui Jiang

2014-01-01

Full Text Available You Gui Wan (YGW is a classic herbal formula in traditional Chinese medicine (TCM used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH. YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01. Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001. YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action.

Partial-Body Irradiation in Patients with Prostate Cancer Treated with IMRT Has Little Effect on the Composition of Serum Proteome.

Science.gov (United States)

Pietrowska, Monika; Jelonek, Karol; Polanska, Joanna; Wojakowska, Anna; Marczak, Lukasz; Chawinska, Ewa; Chmura, Aleksanda; Majewski, Wojciech; Miszczyk, Leszek; Widlak, Piotr

2015-06-30

Partial body irradiation during cancer radiotherapy (RT) induces a response of irradiated tissues that could be observed at the level of serum proteome. Here we aimed to characterize the response to RT in group of patients treated because of prostate cancer. Five consecutive blood samples were collected before, during, and after the end of RT in a group of 126 patients who received definitive treatment with a maximum dose of 76 Gy. Serum peptidome, which was profiled in the 2000-16,000 Da range using MALDI-MS. Serum proteins were identified and quantified using the shotgun LC-MS/MS approach. The majority of changes in serum peptidome were detected between pre-treatment samples and samples collected after 3-4 weeks of RT (~25% of registered peptides changed their abundances significantly), yet the intensity of observed changes was not correlated significantly with the degree of acute radiation toxicity or the volume of irradiated tissues. Furthermore, there were a few serum proteins identified, the abundances of which were different in pre-RT and post-RT samples, including immunity and inflammation-related factors. Observed effects were apparently weaker than in comparable groups of head and neck cancer patients in spite of similar radiation doses and volumes of irradiated tissues in both groups. We concluded that changes observed at the level of serum proteome were low for this cohort of prostate cancer patients, although the specific components involved are associated with immunity and inflammation, and reflect the characteristic acute response of the human body to radiation.

Partial-Body Irradiation in Patients with Prostate Cancer Treated with IMRT Has Little Effect on the Composition of Serum Proteome

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Monika Pietrowska

2015-06-01

Full Text Available Partial body irradiation during cancer radiotherapy (RT induces a response of irradiated tissues that could be observed at the level of serum proteome. Here we aimed to characterize the response to RT in group of patients treated because of prostate cancer. Five consecutive blood samples were collected before, during, and after the end of RT in a group of 126 patients who received definitive treatment with a maximum dose of 76 Gy. Serum peptidome, which was profiled in the 2000–16,000 Da range using MALDI-MS. Serum proteins were identified and quantified using the shotgun LC-MS/MS approach. The majority of changes in serum peptidome were detected between pre-treatment samples and samples collected after 3–4 weeks of RT (~25% of registered peptides changed their abundances significantly, yet the intensity of observed changes was not correlated significantly with the degree of acute radiation toxicity or the volume of irradiated tissues. Furthermore, there were a few serum proteins identified, the abundances of which were different in pre-RT and post-RT samples, including immunity and inflammation-related factors. Observed effects were apparently weaker than in comparable groups of head and neck cancer patients in spite of similar radiation doses and volumes of irradiated tissues in both groups. We concluded that changes observed at the level of serum proteome were low for this cohort of prostate cancer patients, although the specific components involved are associated with immunity and inflammation, and reflect the characteristic acute response of the human body to radiation.

Clinical proteomic analysis of scrub typhus infection.

Science.gov (United States)

Park, Edmond Changkyun; Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Hayoung; Song, Hyun Seok; Jun, Sangmi; Kim, Gun-Hwa; Lee, Chang-Seop; Kim, Seung Il

2018-01-01

Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi , and normal expression was largely rescued by antibiotic treatment. This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

Glyco-centric lectin magnetic bead array (LeMBA − proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals

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Alok K. Shah

2016-06-01

Full Text Available This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA – mass spectrometry techniques, “Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma” [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE and esophageal adenocarcinoma (EAC individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉 using search key “jn7qafftux”. The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Myostatin deficiency but not anti-myostatin blockade induces marked proteomic changes in mouse skeletal muscle.

Science.gov (United States)

Salzler, Robert R; Shah, Darshit; Doré, Anthony; Bauerlein, Roy; Miloscio, Lawrence; Latres, Esther; Papadopoulos, Nicholas J; Olson, William C; MacDonald, Douglas; Duan, Xunbao

2016-07-01

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global Proteome Analysis of Leptospira interrogans

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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...

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

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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.

Proteomic profiling reveals α1-antitrypsin, α1-microglobulin, and clusterin as preeclampsia-related serum proteins in pregnant women.

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Hsu, Te-Yao; Hsieh, T'sang-T'ang; Yang, Kuender D; Tsai, Ching-Chang; Ou, Chia-Yu; Cheng, Bi-Hua; Wong, Yi-Hsun; Hung, Hsuan-Ning; Chou, An-Kuo; Hsiao, Chang-Chun; Lin, Hao

2015-10-01

Preeclampsia is a major cause of mortality in pregnant women but the underlying mechanism remains unclear to date. In this study, we attempted to identify candidate proteins that might be associated with preeclampsia in pregnant women by means of proteomics tools. Differentially expressed proteins in serum samples obtained from pregnant women with severe preeclampsia (n = 8) and control participants (n = 8) were identified using two-dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Additional serum samples from 50 normal and 41 pregnant women with severe preeclampsia were analyzed by immunoassay for validation. Ten protein spots were found to be upregulated significantly in women with severe preeclampsia. These protein spots had the peptide mass fingerprints matched to α1-antitrypsin, α1-microglobulin, clusterin, and haptoglobin. Immunoassays in an independent series of serum samples showed that serum α1-antitrypsin, α1-microglobulin, and clusterin levels of severe preeclampsia patients (n = 41) were significantly higher than those in the normal participants (n = 50; α1-antitrypsin 295.95 ± 50.94 mg/dL vs. 259.31 ± 33.90 mg/dL, p = 0.02; α1-microglobulin 0.029 ± 0.004 mg/mL vs. 0.020 ± 0.004 mg/mL, p proteins by proteomics analysis enables further understanding of the pathophysiology of preeclampsia. Further studies are warranted to investigate the role of these biomarkers in prediction of this disease. Copyright © 2015. Published by Elsevier B.V.

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.

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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.

Migration inhibition of immune mouse spleen cells by serum from x-irradiated tumor-bearing mice

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Moroson, H.

1978-01-01

Tumor-specific antigens of the chemically induced MC 429 mouse fibrosarcoma were detected in a 3 M KCl extract of tumor by the inhibition of migration of specifically immune spleen cells. Using this assay with serum from tumor-bearing mice no tumor antigen was detected in serum of mice bearing small tumors, unless the tumor was exposed to local x irradiation (3000 R) 1 day prior to collection of serum. It was concluded that local x irradiation of tumor caused increased concentration of tumor antigen in the serum. When the tumor was allowed to grow extremely large, with necrosis, then host serum did cause migration inhibition of both nonimmune and immune spleen cells. This migration-inhibition effect was not associated with tumor antigen, but with a nonspecific serum factor

Proteomic profile of serum of pregnant women carring a fetus with Down syndrome using nano uplc Q-tof ms/ms technology.

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López Uriarte, Graciela Arelí; Burciaga Flores, Carlos Horacio; Torres de la Cruz, Víctor Manuel; Medina Aguado, María Magdalena; Gómez Puente, Viviana Maricela; Romero Gutiérrez, Liliana Nayeli; Martínez de Villarreal, Laura Elia

2018-06-01

Prenatal diagnosis of Down syndrome (DS) is based on the calculated risk of maternal age, biochemical and ultrasonographic markers and recently by cfDNA. Differences in proteomic profiles may give an opportunity to find new biomarkers. Characterize proteome of serum of mothers carrying DS fetus. Blood serum samples of three groups of women were obtained, (a) 10 non-pregnant, (b) 10 pregnant with healthy fetus by ultrasound evaluation, (c) nine pregnant with DS fetus. Sample preparation was as follows: Albumin/IgG depletion, desalting, and trypsin digestion; the process was performed in nanoUPLC MS/MS. Data analysis was made with Mass Lynx 4.1 and ProteinLynx Global Server 3.0, peptide and protein recognition by MASCOT algorithm and UNIPROT-Swissprot database. Each group showed different protein profiles. Some proteins were shared between groups. Only sera from pregnant women showed proteins related to immune and clot pathways. Mothers with DS fetus had 42 specific proteins. We found a different serum protein profile in mothers carrying DS fetuses that do not reflect expression of genes in the extra chromosome. Further studies will be necessary to establish the role of these proteins in aneuploid fetus and analyze their possible use as potential biomarkers.

Proteomic analysis of swine serum following highly virulent classical swine fever virus infection

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Guo Huan-cheng

2011-03-01

Full Text Available Abstract Background Classical swine fever virus (CSFV belongs to the genus Pestivirus within the family Flaviviridae. Virulent strains of classical swine fever virus (CSFV cause severe disease in pigs characterized by immunosuppression, thrombocytopenia and disseminated intravascular coagulation, which causes significant economic losses to the pig industry worldwide. Methods To reveal proteomic changes in swine serum during the acute stage of lethal CSFV infection, 5 of 10 pigs were inoculated with the virulent CSFV Shimen strain, the remainder serving as uninfected controls. A serum sample was taken at 3 days post-infection from each swine, at a stage when there were no clinical symptoms other than increased rectal temperatures (≥40°C. The samples were treated to remove serum albumin and immunoglobulin (IgG, and then subjected to two-dimension differential gel electrophoresis. Results Quantitative intensity analysis revealed 17 protein spots showing at least 1.5-fold quantitative alteration in expression. Ten spots were successfully identified by MALDI-TOF MS or LTQ MS. Expression of 4 proteins was increased and 6 decreased in CSFV-infected pigs. Functions of these proteins included blood coagulation, anti-inflammatory activity and angiogenesis. Conclusion These proteins with altered expression may have important implications in the pathogenesis of classical swine fever and provide a clue for identification of biomarkers for classical swine fever early diagnosis.

Serum proteome profiling identifies novel and powerful markers of cystic fibrosis liver disease.

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Timo Rath

Full Text Available BACKGROUND AND AIMS: Cystic Fibrosis associated liver disease (CFLD develops in approximately 30% of CF patients. However, routine sensitive diagnostic tools for CFLD are lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CFLD. METHODS: 45 CF patients were included in the study and received transient elastography. Differential regulation of 220 different serum proteins was assessed in a subgroup of patients with and without CFLD. Most interesting candidate proteins were further quantified and validated by ELISA in the whole patient cohort. To assess a potential relation of biomarker expression to the degree of hepatic fibrosis, serum biomarkers were further determined in 18 HCV patients where liver histology was available. RESULTS: 43 serum proteins differed at least 2-fold in patients with CFLD compared to those without liver disease as identified in proteome profiling. In ELISA quantifications, TIMP-4 and Endoglin were significantly up-regulated in patients with CFLD as diagnosed by clinical guidelines or increased liver stiffness. Pentraxin-3 was significantly decreased in patients with CFLD. Serum TIMP-4 and Endoglin showed highest values in HCV patients with liver cirrhosis compared to those with fibrosis but without cirrhosis. At a cut-off value of 6.3 kPa, transient elastography compassed a very high diagnostic accuracy and specificity for the detection of CFLD. Among the biomarkers, TIMP-4 and Endoglin exhibited a high diagnostic accuracy for CFLD. Diagnostic sensitivities and negative predictive values were increased when elastography and TIMP-4 and Endoglin were combined for the detection of CFLD. CONCLUSIONS: Serum TIMP-4 and Endoglin are increased in CFLD and their expression correlates with hepatic staging. Determination of TIMP-4 and Endoglin together with transient elastography can increase the sensitivity for the non-invasive diagnosis of CFLD.

Serum amyloid A as a prognostic marker in melanoma identified by proteomic profiling.

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Findeisen, Peter; Zapatka, Marc; Peccerella, Teresa; Matzk, Heike; Neumaier, Michael; Schadendorf, Dirk; Ugurel, Selma

2009-05-01

Currently known prognostic serum biomarkers of melanoma are powerful in metastatic disease, but weak in early-stage patients. This study was aimed to identify new prognostic biomarkers of melanoma by serum mass spectrometry (MS) proteomic profiling, and to validate candidates compared with established markers. Two independent sets of serum samples from 596 melanoma patients were investigated. The first set (stage I = 102; stage IV = 95) was analyzed by matrix assisted laser desorption and ionization time of flight (MALDI TOF) MS for biomarkers differentiating between stage I and IV. In the second set (stage I = 98; stage II = 91; stage III = 87; stage IV = 103), the serum concentrations of the candidate marker serum amyloid A (SAA) and the known biomarkers S100B, lactate dehydrogenase, and C reactive protein (CRP) were measured using immunoassays. MALDI TOF MS revealed a peak at m/z 11.680 differentiating between stage I and IV, which could be identified as SAA. High peak intensities at m/z 11.680 correlated with poor survival. In univariate analysis, SAA was a strong prognostic marker in stage I to III (P = .043) and stage IV (P = .000083) patients. Combination of SAA and CRP increased the prognostic impact to P = .011 in early-stage (I to III) patients. Multivariate analysis revealed sex, stage, tumor load, S100B, SAA, and CRP as independent prognostic factors, with an interaction between SAA and CRP. In stage I to III patients, SAA combined with CRP was superior to S100B in predicting patients' progression-free and overall survival. SAA combined with CRP might be used as prognostic serological biomarkers in early-stage melanoma patients, helping to discriminate low-risk patients from high-risk patients needing adjuvant treatment.

Application of serum SELDI proteomic patterns in diagnosis of lung cancer

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Zhou Bin

2005-07-01

Full Text Available Abstract Background Currently, no satisfactory biomarkers are available to screen for lung cancer. Surface-Enhanced Laser Desorption/ionization Time-of- Flight Mass Spectrometry ProteinChip system (SELDI-TOF-MS is one of the currently used techniques to identify biomarkers for cancers. The aim of this study is to explore the application of serum SELDI proteomic patterns to distinguish lung cancer patients from healthy individuals. Methods A total of 208 serum samples, including 158 lung cancer patients and 50 healthy individuals, were randomly divided into a training set (including 11 sera from patients with stages I/II lung cancer, 63 from patients with stages III/IV lung cancer and 20 from healthy controls and a blinded test set (including 43 sera from patients with stages I/II lung cancer, 41 from patients with stages III/IV lung cancer and 30 from healthy controls. All samples were analyzed by SELDI technology. The spectra were generated on weak cation exchange (WCX2 chips, and protein peaks clustering and classification analyses were made using Ciphergen Biomarker Wizard and Biomarker Pattern software, respectively. We additionally determined Cyfra21-1 and NSE in the 208 serum samples included in this study using an electrochemiluminescent immunoassay. Results Five protein peaks at 11493, 6429, 8245, 5335 and 2538 Da were automatically chosen as a biomarker pattern in the training set. When the SELDI marker pattern was tested with the blinded test set, it yielded a sensitivity of 86.9%, a specificity of 80.0% and a positive predictive value of 92.4%. The sensitivities provided by Cyfra21-1 and NSE used individually or in combination were significantly lower than that of the SELDI marker pattern (P P Conclusion These results suggest that serum SELDI protein profiling can distinguish lung cancer patients, especially NSCLC patients, from normal subjects with relatively high sensitivity and specificity, and the SELDI-TOF-MS is a potential tool

Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications.

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Ammerlaan, Wim; Trezzi, Jean-Pierre; Lescuyer, Pierre; Mathay, Conny; Hiller, Karsten; Betsou, Fay

2014-08-01

Formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. Serum and plasma processing protocols were validated for fitness-for-purpose in terms of key downstream endpoints, and this article demonstrates methodology for biospecimen processing method validation. Serum and plasma preparation from human blood was optimized for centrifugation conditions with respect to microparticle counts. Optimal protocols were validated for methodology and reproducibility in terms of acceptance criteria based on microparticle counts, DNA and hemoglobin concentration, and metabolomic and proteomic profiles. These parameters were also used to evaluate robustness for centrifugation temperature (4°C versus room temperature [RT]), deceleration (low, medium, high) and blood stability (after a 2-hour delay). Optimal protocols were 10-min centrifugation for serum and 20-min for plasma at 2000 g, medium brake, RT. Methodology and reproducibility acceptance criteria were met for both protocols except for reproducibility of plasma metabolomics. Overall, neither protocol was robust for centrifugation at 4°C versus RT. RT gave higher microparticles and free DNA yields in serum, and fewer microparticles with less hemolysis in plasma. Overall, both protocols were robust for fast, medium, and low deceleration, with a medium brake considered optimal. Pre-centrifugation stability after a 2-hour delay was seen at both temperatures for hemoglobin concentration and proteomics, but not for microparticle counts. We validated serum and plasma collection methods suitable for downstream protein, metabolite, or free nucleic acid-based applications. Temperature and pre-centrifugation delay can influence analytic results, and laboratories and biobanks should systematically record these conditions in the scope of accreditation.

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

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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

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture.

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Lee, Jong-Seon; Kim, Ju-Hwan; Seo, Young-Seok; Yang, Jung-Bo; Kim, Yong-Il; Kim, Hye-Jin; Lee, Ki-Hwan

2013-09-01

This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P media up to 4 weeks did not affect on embryonic development.

A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

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Stewart Stephen

2009-08-01

Full Text Available Abstract Background Non-alcoholic fatty liver disease (NAFLD has a prevalence of over 20% in Western societies. Affected individuals are at risk of developing both cirrhosis and hepatocellular cancer (HCC. Presently there is no cost effective population based means of identifying cirrhotic individuals and even if there were, our ability to perform HCC surveillance in the at risk group is inadequate. We have performed a pilot proteomic study to assess this as a strategy for serum biomarker detection. Methods 2D Gel electrophoresis was performed on immune depleted sera from 3 groups of patients, namely those with (1 pre-cirrhotic NAFLD (2 cirrhotic NAFLD and (3 cirrhotic NAFLD with co-existing HCC. Five spots differentiating at least one of these three groups were characterised by mass spectroscopy. An ELISA assay was optimised and a cross sectional study assessing one of these serum spots was performed on serum from 45 patients with steatohepatitis related cirrhosis and HCC and compared to 77 patients with histologically staged steatohepatitis. Results Four of the spots identified were apolipoprotein isoforms, the pattern of which was able to differentiate the three groups. The 5th spot, seen in the serum of cirrhotic individuals and more markedly in those with HCC, was identified as CD5 antigen like (CD5L. By ELISA assay, although CD5L was markedly elevated in a number of cirrhotic individuals with HCC, its overall ability to distinguish non-cancer from cancer individuals as determined by AUC ROC analysis was poor. However, serum CD5L was dramatically increased, independently of age, sex, and the presence of necroinflammation, in the serum of individuals with NAFLD cirrhosis relative to those with pre-cirrhotic disease. Conclusion This novel proteomic strategy has identified a number of candidate biomarkers which may have benefit in the surveillance and diagnosis of individuals with chronic liver disease and/or HCC.

Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

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Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

2017-02-15

Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

Exploration of Serum Proteomic Profiling and Diagnostic Model That Differentiate Crohn's Disease and Intestinal Tuberculosis.

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Fenming Zhang

Full Text Available To explore the diagnostic models of Crohn's disease (CD, Intestinal tuberculosis (ITB and the differential diagnostic model between CD and ITB by analyzing serum proteome profiles.Serum proteome profiles from 30 CD patients, 21 ITB patients and 30 healthy controls (HCs were analyzed by using weak cationic magnetic beads combined with MALDI-TOF-MS technique to detect the differentially expressed proteins of serum samples. Three groups were made and compared accordingly: group of CD patients and HCs, group of ITB patients and HCs, group of CD patients and ITB patients. Wilcoxon rank sum test was used to screen the ten most differentiated protein peaks (P < 0.05. Genetic algorithm combining with support vector machine (SVM was utilized to establish the optimal diagnostic models for CD, ITB and the optimal differential diagnostic model between CD and ITB. The predictive effects of these models were evaluated by Leave one out (LOO cross validation method.There were 236 protein peaks differently expressed between group of CD patients and HCs, 305 protein peaks differently expressed between group of ITB patients and HCs, 332 protein peaks differently expressed between group of CD patients and ITB patients. Ten most differentially expressed peaks were screened out between three groups respectively (P < 0.05 to establish diagnostic models and differential diagnostic model. A diagnostic model comprising of four protein peaks (M/Z 4964, 3029, 2833, 2900 can well distinguish CD patients and HCs, with a specificity and sensitivity of 96.7% and 96.7% respectively. A diagnostic model comprising four protein peaks (M/Z 3030, 2105, 2545, 4210 can well distinguish ITB patients and HCs, with a specificity and sensitivity of 93.3% and 95.2% respectively. A differential diagnostic model comprising three potential biomarkers protein peaks (M/Z 4267, 4223, 1541 can well distinguish CD patients and ITB patients, with a specificity and sensitivity of 76.2% and 80

Novel TIA biomarkers identified by mass spectrometry-based proteomics.

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George, Paul M; Mlynash, Michael; Adams, Christopher M; Kuo, Calvin J; Albers, Gregory W; Olivot, Jean-Marc

2015-12-01

Transient ischemic attacks remain a clinical diagnosis with significant variability between physicians. Finding reliable biomarkers to identify transient ischemic attacks would improve patient care and optimize treatment. Our aim is to identify novel serum TIA biomarkers through the use of mass spectroscopy-based proteomics. Patients with transient neurologic symptoms were prospectively enrolled. Mass spectrometry-based proteomics, an unbiased method to identify candidate proteins, was used to test the serum of the patients for biomarkers of cerebral ischemia. Three candidate proteins were found, and serum concentrations of these proteins were measured by enzyme-linked immunosorbent assay in a second cohort of prospectively enrolled patients. The Student's t-test was used for comparison. The Benjamini-Hochberg false discovery rate controlling procedure for multiple comparison adjustments determined significance for the proteomic screen. Patients with transient ischemic attacks (n = 20), minor strokes (n = 15), and controls (i.e. migraine, seizure, n = 12) were enrolled in the first cohort. Ceruloplasmin, complement component C8 gamma (C8γ), and platelet basic protein were significantly different between the ischemic group (transient ischemic attack and minor stroke) and the controls (P = 0·0001, P = 0·00027, P = 0·00105, respectively). A second cohort of patients with transient ischemic attack (n = 22), minor stroke (n = 20), and controls' (n = 12) serum was enrolled. Platelet basic protein serum concentrations were increased in the ischemic samples compared with control (for transient ischemic attack alone, P = 0·019, for the ischemic group, P = 0·046). Ceruloplasmin trended towards increased concentrations in the ischemic group (P = 0·127); no significant difference in C8γ (P = 0·44) was found. Utilizing mass spectrometry-based proteomics, platelet basic protein has been identified as a candidate serum

Development of a sandwich ELISA for the thrombin light chain identified by serum proteome analysis

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Kazuyuki Sogawa

2017-08-01

Full Text Available We previously identified novel biomarker candidates in biliary tract cancer (BTC using serum proteome analysis. Among several candidates, we focused on thrombin light chain which is a 4204 Da peptide as the most promising biomarker for BTC. To move thrombin light chain toward potential diagnostic use, we developed an enzyme immunoassay that enables to measure serum thrombin light chain levels.Both one monoclonal antibody specific to the N-termini and one polyclonal antibody were used to develop a sandwich ELISA for thrombin light chain. The assay was evaluated by comparing the results with those obtained by the ClinProt™ system. Serum samples were obtained from 20 patients with BTC, 20 patients with BBTDs and 20 HVs using the ClinProt™ system and ELISA.The results of the established ELISA showed a positive correlation with the findings by ClinProt™ system (slope=0.3386, intercept=34.901, r2=0.9641. The performance of the ELISA was satisfactory in terms of recovery (97.9–102.5% and within-run (1.5–4.8% and between-day (1.9–6.7% reproducibility. Serum thrombin light chain levels were significantly greater in BTC (176.5±47.2 ng/mL than in BBTDs (128.6±17.4 ng/mL and HVs (127.6±16.0 ng/mL (p<0.001.The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum thrombin light chain levels in various cancers. Keywords: Thrombin light chain, Biliary tract cancer, Sandwich ELISA, Serum biomarker

Dynamic changes in the mouse skeletal muscle proteome during denervation-induced atrophy

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Franziska Lang

2017-07-01

Full Text Available Loss of neuronal stimulation enhances protein breakdown and reduces protein synthesis, causing rapid loss of muscle mass. To elucidate the pathophysiological adaptations that occur in atrophying muscles, we used stable isotope labelling and mass spectrometry to quantify protein expression changes accurately during denervation-induced atrophy after sciatic nerve section in the mouse gastrocnemius muscle. Additionally, mice were fed a stable isotope labelling of amino acids in cell culture (SILAC diet containing 13C6-lysine for 4, 7 or 11 days to calculate relative levels of protein synthesis in denervated and control muscles. Ubiquitin remnant peptides (K-ε-GG were profiled by immunoaffinity enrichment to identify potential substrates of the ubiquitin-proteasomal pathway. Of the 4279 skeletal muscle proteins quantified, 850 were differentially expressed significantly within 2 weeks after denervation compared with control muscles. Moreover, pulse labelling identified Lys6 incorporation in 4786 proteins, of which 43 had differential Lys6 incorporation between control and denervated muscle. Enrichment of diglycine remnants identified 2100 endogenous ubiquitination sites and revealed a metabolic and myofibrillar protein diglycine signature, including myosin heavy chains, myomesins and titin, during denervation. Comparative analysis of these proteomic data sets with known atrogenes using a random forest approach identified 92 proteins subject to atrogene-like regulation that have not previously been associated directly with denervation-induced atrophy. Comparison of protein synthesis and proteomic data indicated that upregulation of specific proteins in response to denervation is mainly achieved by protein stabilization. This study provides the first integrated analysis of protein expression, synthesis and ubiquitin signatures during muscular atrophy in a living animal.

Proteomic profiling of occupational medicamentosa-like dermatitis induced by trichloroethylene in serum based on MALDI-TOF MS.

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Liu, Wei; Hong, Wen-Xu; Zhang, Yanfang; Huang, Peiwu; Yang, Xifei; Ren, Xiaohu; Huang, Haiyan; Liu, Jianjun

2015-11-01

Trichloroethylene (TCE) has long been well known as a major pollutant that affects both occupational and general environments. Occupational medicamentosa-like dermatitis induced by TCE (OMLDT) is an autoimmune disease, which has become one of the critical occupational health issues in China. In this study, we analyzed 18 OMLDT patients and 29 professional TCE contact people on serum proteomic analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and ClinProTools bioinformatics software. The intensities of 35 protein/peptide peaks were significantly different between TCE contact controls and OMLDT patients. A pattern of six peaks (m/z 1,450.33, 1,866.16, 3,262.39, 4,109.55, 5,064.85 and 5,956.57) were selected to construct a diagnostic model to discriminate the OMLDT patients from controls with sensitivity and specificity of both 93.8 %. Our findings provide an alternative proteomic approach to differentiate the OMLDT patients from TCE contact workers with high sensitivity and high specificity, which will be of potential value in clinical diagnosis for occupational disease.

Serum Proteome Signature of Radiation Response: Upregulation of Inflammation-Related Factors and Downregulation of Apolipoproteins and Coagulation Factors in Cancer Patients Treated With Radiation Therapy—A Pilot Study

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Widlak, Piotr, E-mail: widlak@io.gliwice.pl [Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice (Poland); Jelonek, Karol; Wojakowska, Anna; Pietrowska, Monika [Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice (Poland); Polanska, Joanna [Institute of Automatics Control, Silesian University of Technology, Gliwice (Poland); Marczak, Łukasz [Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Poznan (Poland); Miszczyk, Leszek; Składowski, Krzysztof [Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice (Poland)

2015-08-01

Purpose: Ionizing radiation affects the proteome of irradiated cells and tissue, yet data concerning changes induced during radiation therapy (RT) in human blood are fragmentary and inconclusive. We aimed to identify features of serum proteome and associated processes involved in response to partial body irradiation during cancer treatment. Methods and Materials: Twenty patients with head and neck squamous cell cancer (HNSCC) and 20 patients with prostate cancer received definitive intensity modulated RT. Blood samples were collected before RT, just after RT, and 1 month after the end of RT. Complete serum proteome was analyzed in individual samples, using a shotgun liquid chromatography-tandem mass spectrometry approach which allowed identification of approximately 450 proteins. Approximately 100 unique proteins were quantified in all samples after exclusion of immunoglobulins, and statistical significance of differences among consecutive samples was assessed. Processes associated with quantified proteins and their functional interactions were predicted using gene ontology tools. Results: RT-induced changes were marked in the HNSCC patient group: 22 upregulated and 33 downregulated proteins were detected in post-RT sera. Most of the changes reversed during follow-up, yet levels of some proteins remained affected 1 month after the end of RT. RT-upregulated proteins were associated with acute phase, inflammatory response, and complement activation. RT-downregulated proteins were associated with transport and metabolism of lipids (plasma apolipoproteins) and blood coagulation. RT-induced changes were much weaker in prostate cancer patients, which corresponded to differences in acute radiation toxicity observed in both groups. Nevertheless, general patterns of RT-induced sera proteome changes were similar in both of the groups of cancer patients. Conclusions: In this pilot study, we proposed to identify a molecular signature of radiation response, based on specific

Serum proteomic patterns of patients with non-small cell lung cancer treated by radiochemotherapy

International Nuclear Information System (INIS)

Li Xianglan; You Qingshan; Yang Yanmei; Ma Yuyan; Tang Yali; Cai Huilong

2007-01-01

Objective:To detect the serum proteomic patterns of patients with non-small cell lung (NSCLC) treated with radiochemotherapy by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) protein chip array techniques, and to screen differential expression protein and observe the changes between the patterns before and after the treatment. Methods: SELDI-TOF-MS and CM-10 protein chips were used to detect the serum proteomic patterns of 35 healthy persons (normal control) and 35 patients with NSCLC before radiochemotherapy. Twenty-six out of the 35 patients after the treatment were also studied. BioMarker Wizard 3.01 and BioMarker Pattern System 5. 01 were used in combination to analyze the data and to develop diagnostic models. Results: Sixteen differential expression protein peaks from a total of 251 protein peaks were automatically chosen, including 8 high expressions and 8 low expressions in patients with NSCLC. Of the 16 protein peaks, 6 protein peak patterns ( M 2 572.1, M 2 885.8, M 3 870.4, M 4 161.4, M 5 739.7 and M 8 164.3 mass/charge ratio [ m/z] ) were observed in model that could be used to distinguish lung cancer' from non-cancer diseases. The sensitivity and specificity results were 91% (32/35)and 83% (29/35). When the SELDI marker pattern was tested with the blinded test set, the sensitivity and specificity were 80% (28/35) and 71% (25/35). The 16 differential expression protein peaks of patients before and after the treatment were obviously different. But the peaks of patients after the treatment trended to those of the normal control. Of the 16 protein peaks, M 2 572.1, M 2 885.8, M 4 664.78, M 9 228.39 and M 9 396.42 were significantly changed. Conclusions: SELDI-TOF-MS is possibly significant for screening differential expression proteins and assessing the treatment efficacy and prognosis of patients, which needs to be demonstrated by further study. (authors)

Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome

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Rahman Hazir

2011-11-01

Full Text Available Abstract Background The effects of fetal calf serum (FCS heat inactivation and bacterial lipopolysaccharide (LPS contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM cells were grown in FCS, either non-heated, or heat inactivated, having low ( Results A total of four proteins (EIF3M, PRS7, PSB4, and SNAPA were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE as compared to cells grown in media with non-heated FCS (NHE. Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1. Conclusion The present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.

The use of urinary proteomics in the assessment of suitability of mouse models for ageing.

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Nkuipou-Kenfack, Esther; Schanstra, Joost P; Bajwa, Seerat; Pejchinovski, Martin; Vinel, Claire; Dray, Cédric; Valet, Philippe; Bascands, Jean-Loup; Vlahou, Antonia; Koeck, Thomas; Borries, Melanie; Busch, Hauke; Bechtel-Walz, Wibke; Huber, Tobias B; Rudolph, Karl L; Pich, Andreas; Mischak, Harald; Zürbig, Petra

2017-01-01

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8-96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing.

Standardized Profiling of The Membrane-Enriched Proteome of Mouse Dorsal Root Ganglia (DRG) Provides Novel Insights Into Chronic Pain.

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Rouwette, Tom; Sondermann, Julia; Avenali, Luca; Gomez-Varela, David; Schmidt, Manuela

2016-06-01

Chronic pain is a complex disease with limited treatment options. Several profiling efforts have been employed with the aim to dissect its molecular underpinnings. However, generated results are often inconsistent and nonoverlapping, which is largely because of inherent technical constraints. Emerging data-independent acquisition (DIA)-mass spectrometry (MS) has the potential to provide unbiased, reproducible and quantitative proteome maps - a prerequisite for standardization among experiments. Here, we designed a DIA-based proteomics workflow to profile changes in the abundance of dorsal root ganglia (DRG) proteins in two mouse models of chronic pain, inflammatory and neuropathic. We generated a DRG-specific spectral library containing 3067 DRG proteins, which enables their standardized quantification by means of DIA-MS in any laboratory. Using this resource, we profiled 2526 DRG proteins in each biological replicate of both chronic pain models and respective controls with unprecedented reproducibility. We detected numerous differentially regulated proteins, the majority of which exhibited pain model-specificity. Our approach recapitulates known biology and discovers dozens of proteins that have not been characterized in the somatosensory system before. Functional validation experiments and analysis of mouse pain behaviors demonstrate that indeed meaningful protein alterations were discovered. These results illustrate how the application of DIA-MS can open new avenues to achieve the long-awaited standardization in the molecular dissection of pathologies of the somatosensory system. Therefore, our findings provide a valuable framework to qualitatively extend our understanding of chronic pain and somatosensation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Serum protein identification and quantification of the corona of 5, 15 and 80 nm gold nanoparticles

International Nuclear Information System (INIS)

Schäffler, Martin; Semmler-Behnke, Manuela; Takenaka, Shinji; Wenk, Alexander; Schleh, Carsten; Johnston, Blair D; Kreyling, Wolfgang G; Sarioglu, Hakan; Hauck, Stefanie M

2013-01-01

When nanoparticles (NP) enter the body they come into contact with body fluids containing proteins which can adsorb to their surface. These proteins may influence the NP interactions with the biological vicinity, eventually determining their biological fate inside the body. Adsorption of the most abundantly binding proteins was studied after an in vitro 24 hr incubation of monodisperse, negatively charged 5, 15 and 80 nm gold spheres (AuNP) in mouse serum by a two-step analysis: proteomic protein identification and quantitative protein biochemistry. The adsorbed proteins were separated from non-adsorbed proteins by centrifugation and gel electrophoresis and identified using a MALDI-TOF-MS-Proteomics-Analyzer. Quantitative analysis of proteins in gel bands by protein densitometry, required the focus on predominantly binding serum proteins. Numerous proteins adsorbed to the AuNP depending on their size, e.g. apolipoproteins or complement C3. The qualitative and quantitative amount of adsorbed proteins differed between 5, 15 and 80 nm AuNP. Band intensities of adsorbed proteins decreased with increasing AuNP sizes based not only on their mass but also on their surface area. Summarizing, the AuNP surface is covered with serum proteins containing transport and immune related proteins among others. Hence, protein binding depends on the size, surface area and curvature of the AuNP. (paper)

Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome

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Rebecca Wangen

2018-01-01

Full Text Available Acute myeloid leukemia (AML primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS. The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1 stored in media containing bovine serum and dimethyl sulfoxide (DMSO; (2 stored as dried cell pellets; and (3 stored as cell lysates in 4% sodium dodecyl sulfate (SDS. MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.

Application of serum SELDI proteomic patterns in diagnosis of lung cancer

International Nuclear Information System (INIS)

Yang, Shuan-ying; Xiao, Xue-yuan; Zhang, Wang-gang; Zhang, Li-juan; Zhang, Wei; Zhou, Bin; Chen, Guoan; He, Da-cheng

2005-01-01

Currently, no satisfactory biomarkers are available to screen for lung cancer. Surface-Enhanced Laser Desorption/ionization Time-of- Flight Mass Spectrometry ProteinChip system (SELDI-TOF-MS) is one of the currently used techniques to identify biomarkers for cancers. The aim of this study is to explore the application of serum SELDI proteomic patterns to distinguish lung cancer patients from healthy individuals. A total of 208 serum samples, including 158 lung cancer patients and 50 healthy individuals, were randomly divided into a training set (including 11 sera from patients with stages I/II lung cancer, 63 from patients with stages III/IV lung cancer and 20 from healthy controls) and a blinded test set (including 43 sera from patients with stages I/II lung cancer, 41 from patients with stages III/IV lung cancer and 30 from healthy controls). All samples were analyzed by SELDI technology. The spectra were generated on weak cation exchange (WCX2) chips, and protein peaks clustering and classification analyses were made using Ciphergen Biomarker Wizard and Biomarker Pattern software, respectively. We additionally determined Cyfra21-1 and NSE in the 208 serum samples included in this study using an electrochemiluminescent immunoassay. Five protein peaks at 11493, 6429, 8245, 5335 and 2538 Da were automatically chosen as a biomarker pattern in the training set. When the SELDI marker pattern was tested with the blinded test set, it yielded a sensitivity of 86.9%, a specificity of 80.0% and a positive predictive value of 92.4%. The sensitivities provided by Cyfra21-1 and NSE used individually or in combination were significantly lower than that of the SELDI marker pattern (P < 0.005 or 0.05, respectively). Based on the results of the test set, we found that the SELDI marker pattern showed a sensitivity of 91.4% in the detection of non-small cell lung cancers (NSCLC), which was significantly higher than that in the detection of small cell lung cancers (P < 0.05); The

Serum protein profiling and proteomics in autistic spectrum disorder using magnetic bead-assisted mass spectrometry.

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Taurines, Regina; Dudley, Edward; Conner, Alexander C; Grassl, Julia; Jans, Thomas; Guderian, Frank; Mehler-Wex, Claudia; Warnke, Andreas; Gerlach, Manfred; Thome, Johannes

2010-04-01

The pathophysiology of autistic spectrum disorder (ASD) is not fully understood and there are no diagnostic or predictive biomarkers. Proteomic profiling has been used in the past for biomarker research in several non-psychiatric and psychiatric disorders and could provide new insights, potentially presenting a useful tool for generating such biomarkers in autism. Serum protein pre-fractionation with C8-magnetic beads and protein profiling by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-ToF-MS) were used to identify possible differences in protein profiles in patients and controls. Serum was obtained from 16 patients (aged 8-18) and age-matched controls. Three peaks in the MALDI-ToF-MS significantly differentiated the ASD sample from the control group. Sub-grouping the ASD patients into children with and without comorbid Attention Deficit and Hyperactivity Disorder, ADHD (ASD/ADHD+ patients, n = 9; ASD/ADHD- patients, n = 7), one peak distinguished the ASD/ADHD+ patients from controls and ASD/ADHD- patients. Our results suggest that altered protein levels in peripheral blood of patients with ASD might represent useful biomarkers for this devastating psychiatric disorder.

Proteomic profiling of renal allograft rejection in serum using magnetic bead-based sample fractionation and MALDI-TOF MS.

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Sui, Weiguo; Huang, Liling; Dai, Yong; Chen, Jiejing; Yan, Qiang; Huang, He

2010-12-01

Proteomics is one of the emerging techniques for biomarker discovery. Biomarkers can be used for early noninvasive diagnosis and prognosis of diseases and treatment efficacy evaluation. In the present study, the well-established research systems of ClinProt Micro solution incorporated unique magnetic bead sample preparation technology, which, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), have become very successful in bioinformatics due to its outstanding performance and reproducibility for discovery disease-related biomarker. We collected fasting blood samples from patients with biopsy-confirmed acute renal allograft rejection (n = 12), chronic rejection (n = 12), stable graft function (n = 12) and also from healthy volunteers (n = 13) to study serum peptidome patterns. Specimens were purified with magnetic bead-based weak cation exchange chromatography and analyzed with a MALDI-TOF mass spectrometer. The results indicated that 18 differential peptide peaks were selected as potential biomarkers of acute renal allograft rejection, and 6 differential peptide peaks were selected as potential biomarkers of chronic rejection. A Quick Classifier Algorithm was used to set up the classification models for acute and chronic renal allograft rejection. The algorithm models recognize 82.64% of acute rejection and 98.96% of chronic rejection episodes, respectively. We were able to identify serum protein fingerprints in small sample sizes of recipients with renal allograft rejection and establish the models for diagnosis of renal allograft rejection. This preliminary study demonstrated that proteomics is an emerging tool for early diagnosis of renal allograft rejection and helps us to better understand the pathogenesis of disease process.

Hippocampal transcriptomic and proteomic alterations in the BTBR mouse model of autism spectrum disorder

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Caitlin M Daimon

2015-11-01

Full Text Available Autism spectrum disorders (ASD are complex heterogeneous neurodevelopmental disorders of an unclear etiology, and no cure currently exists. Prior studies have demonstrated that the black and tan, brachyury (BTBR T+ Itpr3tf/J mouse strain displays a behavioral phenotype with ASD-like features. BTBR T+ Itpr3tf/J mice (referred to simply as BTBR display deficits in social functioning, lack of communication ability, and engagement in stereotyped behavior. Despite extensive behavioral phenotypic characterization, little is known about the genes and proteins responsible for the presentation of the ASD-like phenotype in the BTBR mouse model. In this study, we employed bioinformatics techniques to gain a wide-scale understanding of the transcriptomic and proteomic changes associated with the ASD-like phenotype in BTBR mice. We found a number of genes and proteins to be significantly altered in BTBR mice compared to C57BL/6J (B6 control mice controls such as BDNF, Shank3, and ERK1, which are highly relevant to prior investigations of ASD. Furthermore, we identified distinct functional pathways altered in BTBR mice compared to B6 controls that have been previously shown to be altered in both mouse models of ASD, some human clinical populations, and have been suggested as a possible etiological mechanism of ASD, including axon guidance and regulation of actin cytoskeleton. In addition, our wide-scale bioinformatics approach also discovered several previously unidentified genes and proteins associated with the ASD phenotype in BTBR mice, such as Caskin1, suggesting that bioinformatics could be an avenue by which novel therapeutic targets for ASD are uncovered. As a result, we believe that informed use of synergistic bioinformatics applications represents an invaluable tool for elucidating the etiology of complex disorders like ASD.

Proteomic approaches in cancer risk and response assessment.

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Petricoin, Emanuel F; Liotta, Lance A

2004-02-01

Proteomics is more than just a list-generating exercise where increases or decreases in protein expression are identified. Proteomic technologies will ultimately characterize information-flow through the protein circuitry that interconnects the extracellular microenvironment to the serum or plasma macroenvironment through intracellular signaling systems and their control of gene transcription. The nature of this information can be a cause or a consequence of disease processes and how patients respond to therapy. Analysis of human cancer as a model for how proteomics can have an impact at the bedside can take advantage of several promising new proteomic technologies. These technologies are being developed for early detection and risk assessment, therapeutic targeting and patient-tailored therapy.

iTRAQ-Based Proteomics Analysis of Serum Proteins in Wistar Rats Treated with Sodium Fluoride: Insight into the Potential Mechanism and Candidate Biomarkers of Fluorosis

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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.

Clinical proteomics: Applications for prostate cancer biomarker discovery and detection.

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Petricoin, Emanuel F; Ornstein, David K; Liotta, Lance A

2004-01-01

The science of proteomics comprises much more than simply generating lists of proteins that change in expression as a cause of or consequence of pathophysiology. The goal of proteomics should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. Serum proteomic pattern diagnostics is a new type of proteomic concept in which patterns of ion signatures generated from high dimensional mass spectrometry data are used as diagnostic classifiers. This recent approach has exciting potential for clinical utility of diagnostic patterns because low molecular weight metabolites, peptides, and protein fragments may have higher accuracy than traditional biomarkers of cancer detection. Intriguingly, we now have discovered that this diagnostic information exists in a bound state, complexed with circulating highly abundant carrier proteins. These diagnostic fragments may one day be harvested by circulating nanoparticles, designed to absorb, enrich, and amplify the repertoire of diagnostic biomarkers generated-even at the critical, initial stages of carcinogenesis. Copyright 2004 Elsevier Inc.

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

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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.

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

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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.

Application of bioinformatics to optimization of serum proteome in ...

African Journals Online (AJOL)

SAM

2014-05-14

OSCC) from oral leukoplakia ... study the sera proteomes of 32 healthy volunteers, 6 patients with oral mucosa leukoplakia, 28 OSCC patients, and 8 .... American Ciphergen SELDI Protein Biology System II plus (PBS. II plus) and ...

Serum proteomic changes after randomized prolonged erythropoietin treatment and/or endurance training: detection of novel biomarkers.

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Britt Christensen

Full Text Available Despite implementation of the biological passport to detect erythropoietin abuse, a need for additional biomarkers remains. We used a proteomic approach to identify novel serum biomarkers of prolonged erythropoiesis-stimulating agent (ESA exposure (Darbepoietin-α and/or aerobic training.Thirty-six healthy young males were randomly assigned to the following groups: Sedentary-placebo (n = 9, Sedentary-ESA (n = 9, Training-placebo (n = 10, or Training-ESA (n = 8. They were treated with placebo/Darbepoietin-α subcutaneously once/week for 10 weeks followed by a 3-week washout period. Training consisted of supervised biking 3/week for 13 weeks at the highest possible intensity. Serum was collected at baseline, week 3 (high dose Darbepoietin-α, week 10 (reduced dose Darbepoietin-α, and after a 3-week washout period.Serum proteins were separated according to charge and molecular mass (2D-gel electrophoresis. The identity of proteins from spots exhibiting altered intensity was determined by mass spectrometry.Six protein spots changed in response to Darbepoietin-α treatment. Comparing all 4 experimental groups, two protein spots (serotransferrin and haptoglobin/haptoglobin related protein showed a significant response to Darbepoietin-α treatment. The haptoglobin/haptoglobin related protein spot showed a significantly lower intensity in all subjects in the training-ESA group during the treatment period and increased during the washout period.An isoform of haptoglobin/haptoglobin related protein could be a new anti-doping marker and merits further research.ClinicalTrials.gov NCT01320449.

A novel proteomic biomarker panel as a diagnostic tool for patients with ovarian cancer

DEFF Research Database (Denmark)

Høgdall, Claus; Fung, Eric T; Christensen, Ib J

2011-01-01

Previous reports have shown that the proteomic markers apolipoprotein A1, hepcidin, transferrin, inter-alpha trypsin IV internal fragment, transthyretin, connective-tissue activating protein 3 and beta-2 microglobulin may discriminate between a benign pelvic mass and ovarian cancer (OC). The aim...... was to determine if these serum proteomic biomarkers alone as well as in combination with age and serum CA125, could be helpful in triage of women with a pelvic mass....

Analysis of pig serum proteins based on shotgun liquid ...

African Journals Online (AJOL)

Recent advances in proteomics technologies have opened up significant opportunities for future applications. We used shotgun liquid chromatography, coupled with tandem mass spectrometry (LC-MS/MS) to determine the proteome profile of healthy pig serum. Samples of venous blood were collected and subjected to ...

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

DEFF Research Database (Denmark)

Albrethsen, J.; Kaas, A.; Schonle, E.

2009-01-01

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably...... be examined for potential bias between sample groups. S ELDI-TOF MS protein profiling was used for preliminary evaluation of a biological-bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000......-2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected...

freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

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Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

2015-01-01

Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

Differential Peripheral Proteomic Biosignature of Fluoxetine Response in a Mouse Model of Anxiety/Depression

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Indira Mendez-David

2017-08-01

Full Text Available The incorporation of peripheral biomarkers in the treatment of major depressive disorders (MDD could improve the efficiency of treatments and increase remission rate. Peripheral blood mononuclear cells (PBMCs represent an attractive biological substrate allowing the identification of a drug response signature. Using a proteomic approach with high-resolution mass spectrometry, the present study aimed to identify a biosignature of antidepressant response (fluoxetine, a Selective Serotonin Reuptake Inhibitor in PBMCs in a mouse model of anxiety/depression. Following determination of an emotionality score, using complementary behavioral analysis of anxiety/depression across three different tests (Elevated Plus Maze, Novelty Suppressed Feeding, Splash Test, we showed that a 4-week corticosterone treatment (35 μg/ml, CORT model in C57BL/6NTac male mice induced an anxiety/depressive-like behavior. Then, chronic fluoxetine treatment (18 mg/kg/day for 28 days in the drinking water reduced corticosterone-induced increase in emotional behavior. However, among 46 fluoxetine-treated mice, only 30 of them presented a 50% decrease in emotionality score, defining fluoxetine responders (CORT/Flx-R. To determine a peripheral biological signature of fluoxetine response, proteomic analysis was performed from PBMCs isolated from the “most” affected corticosterone/vehicle (CORT/V, corticosterone/fluoxetine responders and non-responders (CORT/Flx-NR animals. In comparison to CORT/V, a total of 263 proteins were differently expressed after fluoxetine exposure. Expression profile of these proteins showed a strong similarity between CORT/Flx-R and CORT/Flx-NR (R = 0.827, p < 1e-7. Direct comparison of CORT/Flx-R and CORT/Flx-NR groups revealed 100 differently expressed proteins, representing a combination of markers associated either with the maintenance of animals in a refractory state, or associated with behavioral improvement. Finally, 19 proteins showed a

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

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Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

2012-04-06

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

Proteomic and transcriptomic studies of HBV-associated liver fibrosis of an AAV-HBV-infected mouse model.

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Kan, Fangming; Ye, Lei; Yan, Tao; Cao, Jiaqi; Zheng, Jianhua; Li, Wuping

2017-08-22

Human hepatitis B virus (HBV) infection is an important public health issue in the Asia-Pacific region and is associated with chronic hepatitis, liver fibrosis, cirrhosis and even liver cancer. However, the underlying mechanisms of HBV-associated liver fibrosis remain incompletely understood. In the present study, proteomic and transcriptomic approaches as well as biological network analyses were performed to investigate the differentially expressed molecular signature and key regulatory networks that were associated with HBV-mediated liver fibrosis. RNA sequencing and 2DE-MALDI-TOF/TOF were performed on liver tissue samples obtained from HBV-infected C57BL/6 mouse generated via AAV8-HBV virus. The results showed that 322 genes and 173 proteins were differentially expressed, and 28 HBV-specific proteins were identified by comprehensive proteomic and transcriptomic analysis. GO analysis indicated that the differentially expressed proteins were predominantly involved in oxidative stress, which plays a key role in HBV-related liver fibrosis. Importantly, CAT, PRDX1, GSTP1, NXN and BLVRB were shown to be associated with oxidative stress among the differentially expressed proteins. The most striking results were validated by Western blot and RT-qPCR. The RIG-I like receptor signaling pathway was found to be the major signal pathway that changed during HBV-related fibrosis. This study provides novel insights into HBV-associated liver fibrosis and reveals the significant role of oxidative stress in liver fibrosis. Furthermore, CAT, BLVRB, NXN, PRDX1, and IDH1 may be candidates for detection of liver fibrosis or therapeutic targets for the treatment of liver fibrosis.

Sherlock Holmes and the proteome--a detective story.

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Righetti, Pier Giorgio; Boschetti, Egisto

2007-02-01

The performance of a hexapeptide ligand library in capturing the 'hidden proteome' is illustrated and evaluated. This library, insolubilized on an organic polymer and available under the trade name 'Equalizer Bead Technology', acts by capturing all components of a given proteome, by concentrating rare and very rare proteins, and simultaneously diluting the abundant ones. This results in a proteome of 'normalized' relative abundances, amenable to analysis by MS and any other analytical tool. Examples are given of analysis of human urine and serum, as well as cell and tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae extracts. Another important application is impurity tracking and polishing of recombinant DNA products, especially biopharmaceuticals meant for human consumption.

Serum Proteome Changes in Healthy Subjects with Different Genotypes of NOS1AP in the Chinese Population

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Feng Jiang

2013-01-01

Full Text Available Type 2 diabetes and its chronic complications have become a worldwide epidemic nowadays. However, its molecular mechanism is still unknown. We have previously identified a novel variant rs12742393 of NOS1AP for type 2 diabetes susceptibility in the Chinese population. In this study, we analyzed the total serum profiling among three genotypes of rs12742393 to discover potential crosstalk under the variant and the disease through proteomic analyses for the first time. We used OFFGEL peptide fractionation, LC-MS/MS analysis, and label-free quantification to profile the fasting human serum samples of the genotypes in rs12742393 (n=4, for CC, AC, and AA, resp.. Four proteins were identified, including apoA4, alpha1-ACT, HABP2, and keratin 10, with blood levels changed significantly between CC and AA homozygotes of rs12742393. Compared with AA group, the levels of apoA4 increased (P=0.000265, whereas the concentration of alpha1-ACT, HABP2, and keratin 10 decreased in CC group (P=0.011116, 0.021175, and 0.015661, resp.. Then we selected additional fasting serum samples for ELISA and western blot validation. However, no significant differences were identified by neither ELISA nor western blot (P>0.05. The protein profiling changes between the genotypes of rs12742393 indicated that this SNP might play a role in the development of type 2 diabetes.

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

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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.

Increased circulating resistin levels in early-onset breast cancer patients of normalbody mass index correlate with lymphnode negative involvement and longerdisease free survival: a multi-center POSH cohort serum proteomics study

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Zeidan, Bashar; Manousopoulou, Antigoni; Garay Baquero, Diana J.; White, Cory H.; Larkin, Samantha E.T.; Potter, Kathleen N.; Roumeliotis, Theodoros I.; Papachristou, Evangelia K.; Copson, Ellen; Cutress, Ramsey I.; Beers, Stephen A.; Eccles, Diana; Townsend, Paul A.; Garbis, Spiros D.

2018-01-01

BackgroundEarly-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC.MethodsSerum samples from EOBC patients (stages 1–3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-yea...

Combinatorial hexapeptide ligand libraries (ProteoMiner): an innovative fractionation tool for differential quantitative clinical proteomics.

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Hartwig, Sonja; Czibere, Akos; Kotzka, Jorg; Passlack, Waltraud; Haas, Rainer; Eckel, Jürgen; Lehr, Stefan

2009-07-01

Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner). Here, we demonstrate that ProteoMiner can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 microg to 300 microg) of whole E. coli lysate, processed it with ProteoMiner and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner treatment. Therefore, we conclude that the ProteoMiner technology can be used for quantitative analysis of low abundant proteins in complex biological samples.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

DEFF Research Database (Denmark)

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....

HUPO BPP Workshop on Mouse Models for Neurodegeneration--Choosing the right models.

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Hamacher, Michael; Marcus, Katrin; Stephan, Christian; van Hall, Andre; Meyer, Helmut E

2005-09-01

The HUPO Brain Proteome Project met during the 4th Dutch Endo-Neuro-Psycho Meeting in Doorwerth, The Netherlands, on June 1, 2005, in order to discuss appropriate (mouse) models for neurodegenerative diseases as well as to conceptualise sophisticated proteomics analyses strategies. Here, the topics of the meeting are summarised.

Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles.

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Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei; Mackintosh, Samuel G; Tackett, Alan J; Prayaga, Sastry V S; Romeo, Francesco; Shmookler Reis, Robert J; Mehta, Jawahar L

2016-05-01

Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each Phypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual. © 2016 American Heart Association, Inc.

Application of mass spectrometry-based proteomics for biomarker discovery in neurological disorders

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Venugopal Abhilash

2009-01-01

Full Text Available Mass spectrometry-based quantitative proteomics has emerged as a powerful approach that has the potential to accelerate biomarker discovery, both for diagnostic as well as therapeutic purposes. Proteomics has traditionally been synonymous with 2D gels but is increasingly shifting to the use of gel-free systems and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS. Quantitative proteomic approaches have already been applied to investigate various neurological disorders, especially in the context of identifying biomarkers from cerebrospinal fluid and serum. This review highlights the scope of different applications of quantitative proteomics in understanding neurological disorders with special emphasis on biomarker discovery.

Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

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Chin, Mark H.; Qian, Weijun; Wang, Haixing; Petyuk, Vladislav A.; Bloom, Joshua S.; Sforza, Daniel M.; Lacan, Goran; Liu, Dahai; Khan, Arshad H.; Cantor, Rita M.; Bigelow, Diana J.; Melega, William P.; Camp, David G.; Smith, Richard D.; Smith, Desmond J.

2008-02-10

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.

Proteome profiling analysis of human ovarian cancer serum samples

International Nuclear Information System (INIS)

Cognetti, F.; Citro, G.

2009-01-01

Mass Spectrometry represents a powerful tool in cancer research to discovery of potential bio markers through peak identification from serum profiling. By using high resolution MALDITOF and bioinformatic analysis almost 400 serum sample homogeneously distributed between biopsy confirmed ovarian cancer and high risk serum samples were analyzed. Each serum sample run in duplicate and whole serum sample preparation procedure has been performed by Hamilton Star Robot in order to reduce bias and the replicates with a low Pearson coefficient are removed. After automated reverse phase magnetic beads separation the samples were tested in MALDI-TOF

Microwave and magnetic (M2 proteomics of a mouse model of mild traumatic brain injury

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Teresa M. Evans

2014-06-01

Full Text Available Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI. However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave and magnetic (M2 proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP and p < 0.05 for myelin associated glycoprotein (MAG. This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2 and neurofilament light (NEFL expression at 30 days post-injury were directly related to grip strength (p < 0.05. While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M2 proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment.

Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy.

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Carberry, Steven; Brinkmeier, Heinrich; Zhang, Yaxin; Winkler, Claudia K; Ohlendieck, Kay

2013-09-01

Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20-25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein

The Proteomics Big Challenge for Biomarkers and New Drug-Targets Discovery

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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

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients.

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Zhang, Yuan; Chen, Xianqiu; Hu, Yang; Du, Shanshan; Shen, Li; He, Yifan; Zhang, Yuxuan; Zhang, Xia; Li, Huiping; Yung, Rex C

2013-02-11

The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis. Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue. An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

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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.

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

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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.

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients

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Zhang Yuan

2013-02-01

Full Text Available Abstract Background The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis. Methods Serum samples were collected from patients with sarcoidosis (n = 37, and compared to those from patients with tuberculosis (n = 20, other pulmonary diseases (n = 20, and healthy volunteers (n = 20 for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue. Results An unique protein peak of M/Z 3,210 Daltons (Da was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122 of serum amyloid A (SAA. ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p p  Conclusion This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non

PCAS – a precomputed proteome annotation database resource

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Luo Jingchu

2003-11-01

Full Text Available Abstract Background Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources. Results We report here the development of PCAS (ProteinCentric Annotation System as an online resource of pre-computed proteome annotation data. We applied most available motif or domain databases and their analysis methods, including hmmpfam search of HMMs in Pfam, SMART and TIGRFAM, RPS-PSIBLAST search of PSSMs in CDD, pfscan of PROSITE patterns and profiles, as well as PSI-BLAST search of SUPERFAMILY PSSMs. In addition, signal peptide and TM are predicted using SignalP and TMHMM respectively. We mapped SUPERFAMILY and COGs to InterPro, so the motif or domain databases are integrated through InterPro. PCAS displays table summaries of pre-computed data and a graphical presentation of motifs or domains relative to the protein. As of now, PCAS contains human IPI, mouse IPI, and rat IPI, A. thaliana, C. elegans, D. melanogaster, S. cerevisiae, and S. pombe proteome. PCAS is available at http://pak.cbi.pku.edu.cn/proteome/gca.php Conclusion PCAS gives better annotation coverage for model proteomes by employing a wider collection of available algorithms. Besides presenting the most confident annotation data, PCAS also allows customized query so users can inspect statistically less significant boundary information as well. Therefore, besides providing general annotation information, PCAS could be used as a discovery platform. We plan to update PCAS twice a year. We will upgrade PCAS when new proteome annotation algorithms

Transcriptomic and Proteomic Profiling Revealed High Proportions of Odorant Binding and Antimicrobial Defense Proteins in Olfactory Tissues of the House Mouse

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Barbora Kuntová

2018-02-01

Full Text Available Mammalian olfaction depends on chemosensory neurons of the main olfactory epithelia (MOE, and/or of the accessory olfactory epithelia in the vomeronasal organ (VNO. Thus, we have generated the VNO and MOE transcriptomes and the nasal cavity proteome of the house mouse, Mus musculus musculus. Both transcriptomes had low levels of sexual dimorphisms, while the soluble proteome of the nasal cavity revealed high levels of sexual dimorphism similar to that previously reported in tears and saliva. Due to low levels of sexual dimorphism in the olfactory receptors in MOE and VNO, the sex-specific sensing seems less likely to be dependent on receptor repertoires. However, olfaction may also depend on a continuous removal of background compounds from the sites of detection. Odorant binding proteins (OBPs are thought to be involved in this process and in our study Obp transcripts were most expressed along other lipocalins (e.g., Lcn13, Lcn14 and antimicrobial proteins. At the level of proteome, OBPs were highly abundant with only few being sexually dimorphic. We have, however, detected the major urinary proteins MUP4 and MUP5 in males and females and the male-biased central/group-B MUPs that were thought to be abundant mainly in the urine. The exocrine gland-secreted peptides ESP1 and ESP22 were male-biased but not male-specific in the nose. For the first time, we demonstrate that the expression of nasal lipocalins correlates with antimicrobial proteins thus suggesting that their individual variation may be linked to evolvable mechanisms that regulate natural microbiota and pathogens that regularly enter the body along the ‘eyes-nose-oral cavity’ axis.

On the tear proteome of the house mouse (Mus musculus musculus in relation to chemical signalling

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Romana Stopkova

2017-07-01

Full Text Available Mammalian tears are produced by lacrimal glands to protect eyes and may function in chemical communication and immunity. Recent studies on the house mouse chemical signalling revealed that major urinary proteins (MUPs are not individually unique in Mus musculus musculus. This fact stimulated us to look for other sexually dimorphic proteins that may—in combination with MUPs—contribute to a pool of chemical signals in tears. MUPs and other lipocalins including odorant binding proteins (OBPs have the capacity to selectively transport volatile organic compounds (VOCs in their eight-stranded beta barrel, thus we have generated the tear proteome of the house mouse to detect a wider pool of proteins that may be involved in chemical signalling. We have detected significant male-biased (7.8% and female-biased (7% proteins in tears. Those proteins that showed the most elevated sexual dimorphisms were highly expressed and belong to MUP, OBP, ESP (i.e., exocrine gland-secreted peptides, and SCGB/ABP (i.e., secretoglobin families. Thus, tears may have the potential to elicit sex-specific signals in combination by different proteins. Some tear lipocalins are not sexually dimorphic—with MUP20/darcin and OBP6 being good examples—and because all proteins may flow with tears through nasolacrimal ducts to nasal and oral cavities we suggest that their roles are wider than originally thought. Also, we have also detected several sexually dimorphic bactericidal proteins, thus further supporting an idea that males and females may have adopted alternative strategies in controlling microbiota thus yielding different VOC profiles.

ABCD2 identifies a subclass of peroxisomes in mouse adipose tissue

Energy Technology Data Exchange (ETDEWEB)

Liu, Xiaoxi, E-mail: xiaoxi.liu@uky.edu; Liu, Jingjing, E-mail: jingjing.liu0@gmail.com; Lester, Joshua D., E-mail: joshua.lester@uky.edu; Pijut, Sonja S., E-mail: srhee2@uky.edu; Graf, Gregory A., E-mail: Gregory.Graf@uky.edu

2015-01-02

Highlights: • We examined the D2 localization and the proteome of D2-containing compartment in mouse adipose tissue. • We confirmed the presence of D2 on a subcellular compartment that has typical structure as a microperoxisome. • We demonstrated the scarcity of peroxisome markers on D2-containing compartment. • The D2-containing compartment may be a subpopulation of peroxisome in mouse adipose tissue. • Proteomic data suggests potential association between D2-containing compartment and mitochondria and ER. - Abstract: ATP-binding cassette transporter D2 (D2) is an ABC half transporter that is thought to promote the transport of very long-chain fatty acyl-CoAs into peroxisomes. Both D2 and peroxisomes increase during adipogenesis. Although peroxisomes are essential to both catabolic and anabolic lipid metabolism, their function, and that of D2, in adipose tissues remain largely unknown. Here, we investigated the D2 localization and the proteome of D2-containing organelles, in adipose tissue. Centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but deficient in peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with a dense matrix and a diameter of ∼200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment confirmed the scarcity of PEX19 and proteomic profiling revealed the presence of proteins associated with peroxisome, endoplasmic reticulum (ER), and mitochondria. D2 is localized to a distinct class of peroxisomes that lack many peroxisome proteins, and may associate physically with mitochondria and the ER.

Informed-Proteomics: open-source software package for top-down proteomics

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Park, Jungkap; Piehowski, Paul D.; Wilkins, Christopher; Zhou, Mowei; Mendoza, Joshua; Fujimoto, Grant M.; Gibbons, Bryson C.; Shaw, Jared B.; Shen, Yufeng; Shukla, Anil K.; Moore, Ronald J.; Liu, Tao; Petyuk, Vladislav A.; Tolić, Nikola; Paša-Tolić, Ljiljana; Smith, Richard D.; Payne, Samuel H.; Kim, Sangtae

2017-08-07

Top-down proteomics involves the analysis of intact proteins. This approach is very attractive as it allows for analyzing proteins in their endogenous form without proteolysis, preserving valuable information about post-translation modifications, isoforms, proteolytic processing or their combinations collectively called proteoforms. Moreover, the quality of the top-down LC-MS/MS datasets is rapidly increasing due to advances in the liquid chromatography and mass spectrometry instrumentation and sample processing protocols. However, the top-down mass spectra are substantially more complex compare to the more conventional bottom-up data. To take full advantage of the increasing quality of the top-down LC-MS/MS datasets there is an urgent need to develop algorithms and software tools for confident proteoform identification and quantification. In this study we present a new open source software suite for top-down proteomics analysis consisting of an LC-MS feature finding algorithm, a database search algorithm, and an interactive results viewer. The presented tool along with several other popular tools were evaluated using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.

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

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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.

Acute toxicity of functionalized single wall carbon nanotubes: A biochemical, histopathologic and proteomics approach.

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Ahmadi, Homa; Ramezani, Mohammad; Yazdian-Robati, Rezvan; Behnam, Behzad; Razavi Azarkhiavi, Kamal; Hashem Nia, Azadeh; Mokhtarzadeh, Ahad; Matbou Riahi, Maryam; Razavi, Bibi Marjan; Abnous, Khalil

2017-09-25

Recently carbon nanotubes (CNTs) showed promising potentials in different biomedical applications but their safe use in humans and probable toxicities are still challenging. The aim of this study was to determine the acute toxicity of functionalized single walled carbon nanotubes (SWCNTs). In this project, PEGylated and Tween functionalized SWCNTs were prepared. BALB/c mice were randomly divided into nine groups, including PEGylated SWCNTs (75,150μg/mouse) and PEG, Tween80 suspended SWCNTs, Tween 80 and a control group (intact mice). One or 7 days after intravenous injection, the mice were killed and serum and livers were collected. The oxidative stress markers, biochemical and histopathological changes were studied. Subsequently, proteomics approach was used to investigate the alterations of protein expression profiles in the liver. Results showed that there were not any significant differences in malondealdehyde (MDA), glutathione (GSH) levels and biochemical enzymes (ALT and AST) between groups, while the histopathological observations of livers showed some injuries. The results of proteomics analysis revealed indolethylamine N-Methyltransferase (INMT), glycine N-Methyltransferase (GNMT), selenium binding protein (Selenbp), thioredoxin peroxidase (TPx), TNF receptor associated protein 1(Trap1), peroxiredoxin-6 (Prdx6), electron transport flavoprotein (Etf-α), regucalcin (Rgn) and ATP5b proteins were differentially expressed in functionalized SWCNTs groups. Western blot analyses confirmed that the changes in Prdx6 were consistent with 2-DE gel analysis. In summary, acute toxicological study on two functionalized SWCNTs did not show any significant toxicity at selected doses. Proteomics analysis also showed that following exposure to functionalized SWCNTs, the expression of some proteins with antioxidant activity and detoxifying properties were increased in liver tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

Secreted autoantibody repertoires in Sjögren's syndrome and systemic lupus erythematosus: A proteomic approach.

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Al Kindi, Mahmood A; Colella, Alex D; Chataway, Tim K; Jackson, Michael W; Wang, Jing J; Gordon, Tom P

2016-04-01

The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. A novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis, and de novo and database-driven sequencing of V-region proteins by mass spectrometry. Analysis of anti-Ro52/Ro60/La proteomes in primary Sjögren's syndrome (SS) and anti-Sm and anti-ribosomal P proteomes in systemic lupus erythematosus (SLE) has revealed that these antibody responses are dominated by restricted sets of public (shared) clonotypes, consistent with common pathways of production across unrelated individuals. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted mass spectrometry platforms and for tracking and removal of pathogenic clones. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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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

Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow

International Nuclear Information System (INIS)

Kulkarni, Shilpa; Koller, Antonius; Mani, Kartik M.; Wen, Ruofeng; Alfieri, Alan; Saha, Subhrajit; Wang, Jian; Patel, Purvi; Bandeira, Nuno; Guha, Chandan

2016-01-01

Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted

Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow

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Kulkarni, Shilpa [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Koller, Antonius [Proteomics Center, Stony Brook University School of Medicine, Stony Brook, New York (United States); Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, New York (United States); Mani, Kartik M. [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Wen, Ruofeng [Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York (United States); Alfieri, Alan; Saha, Subhrajit [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Wang, Jian [Center for Computational Mass Spectrometry, University of California, San Diego, California (United States); Department of Computer Science and Engineering, University of California, San Diego, California (United States); Patel, Purvi [Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, New York (United States); Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York (United States); Bandeira, Nuno [Center for Computational Mass Spectrometry, University of California, San Diego, California (United States); Department of Computer Science and Engineering, University of California, San Diego, California (United States); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California (United States); Guha, Chandan, E-mail: cguha@montefiore.org [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); and others

2016-11-01

Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted

Immunohistochemical Distribution of Serum Proteins in Living Mouse Heart with In Vivo Cryotechnique

International Nuclear Information System (INIS)

Shi, Liye; Terada, Nobuo; Saitoh, Yurika; Saitoh, Sei; Ohno, Shinichi

2011-01-01

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods

Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

International Nuclear Information System (INIS)

Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F.

1990-01-01

[ 125 I]Iodomouse GH [( 125 I]iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse

Mass spectrometry analysis of hepcidin peptides in experimental mouse models.

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Harold Tjalsma

Full Text Available The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1 and its paralogue Hepcidin-2 (Hep-2 at the peptide level. To this purpose, Fourier transform ion cyclotron resonance (FTICR and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i 3 mouse strains (C57Bl/6; DBA/2 and BABL/c upon stimulation with intravenous iron and LPS, ii homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X mutated mice and double affected mice, and iii mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.

The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse

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Aiping Zhang

2015-01-01

Full Text Available Phosphorodiamidate morpholino oligonucleotides (PMO are used as a promising exon-skipping gene therapy for Duchenne muscular dystrophy (DMD. One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23. We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10 mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg. Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI. The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at days 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO.

Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

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Norelle C. Wildburger

2015-09-01

Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

NeuCode Proteomics Reveals Bap1 Regulation of Metabolism

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Joshua M. Baughman

2016-07-01

Full Text Available We introduce neutron-encoded (NeuCode amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.

Mining novel biomarkers for prognosis of gastric cancer with serum proteomics

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Sui Mei-Hua

2009-09-01

Full Text Available Abstract Background Although gastric caner (GC remains the second cause of cancer-related death, useful biomarkers for prognosis are still unavailable. We present here the attempt of mining novel biomarkers for GC prognosis by using serum proteomics. Methods Sera from 43 GC patients and 41 controls with gastritis as Group 1 and 11 GC patients as Group 2 was successively detected by Surface Enhanced Laser Desorption/ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS with Q10 chip. Peaks were acquired by Ciphergen ProteinChip Software 3.2.0 and analyzed by Zhejiang University-ProteinChip Data Analysis System (ZJU-PDAS. CEA level were evaluated by chemiluminescence immunoassay. Results After median follow-up periods of 33 months, Group 1 with 4 GC patients lost was divided into 20 good-prognosis GC patients (overall survival more than 24 months and 19 poor-prognosis GC patients (no more than 24 months. The established prognosis pattern consisted of 5 novel prognosis biomarkers with 84.2% sensitivity and 85.0% specificity, which were significantly higher than those of carcinoembryonic antigen (CEA and TNM stage. We also tested prognosis pattern blindly in Group 2 with 66.7% sensitivity and 80.0% specificity. Moreover, we found that 4474-Da peak elevated significantly in GC and was associated with advanced stage (III+IV and short survival (p Conclusion We have identified a number of novel biomarkers for prognosis prediction of GC by using SELDI-TOF-MS combined with sophisticated bioinformatics. Particularly, elevated expression of 4474-Da peak showed very promising to be developed into a novel biomarker associated with biologically aggressive features of GC.

Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics.

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Pisanu, Salvatore; Biosa, Grazia; Carcangiu, Laura; Uzzau, Sergio; Pagnozzi, Daniela

2018-08-01

Seven commercial products for human serum depletion/enrichment were tested and compared by shotgun proteomics. Methods were based on four different capturing agents: antibodies (Qproteome Albumin/IgG Depletion kit, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, Top 2 Abundant Protein Depletion Spin Columns, and Top 12 Abundant Protein Depletion Spin Columns), specific ligands (Albumin/IgG Removal), mixture of antibodies and ligands (Albumin and IgG Depletion SpinTrap), and combinatorial peptide ligand libraries (ProteoMiner beads), respectively. All procedures, to a greater or lesser extent, allowed an increase of identified proteins. ProteoMiner beads provided the highest number of proteins; Albumin and IgG Depletion SpinTrap and ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit resulted the most efficient in albumin removal; Top 2 and Top 12 Abundant Protein Depletion Spin Columns decreased the overall immunoglobulin levels more than other procedures, whereas specifically gamma immunoglobulins were mostly removed by Albumin and IgG Depletion SpinTrap, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, and Top 2 Abundant Protein Depletion Spin Columns. Albumin/IgG Removal, a resin bound to a mixture of protein A and Cibacron Blue, behaved less efficiently than the other products. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

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Ihnatko, R; Post, C; Blomqvist, A

2013-10-01

Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Integration of Serum Protein Biomarker and Tumor Associated Autoantibody Expression Data Increases the Ability of a Blood-Based Proteomic Assay to Identify Breast Cancer.

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Meredith C Henderson

Full Text Available Despite significant advances in breast imaging, the ability to accurately detect Breast Cancer (BC remains a challenge. With the discovery of key biomarkers and protein signatures for BC, proteomic technologies are currently poised to serve as an ideal diagnostic adjunct to imaging. Research studies have shown that breast tumors are associated with systemic changes in levels of both serum protein biomarkers (SPB and tumor associated autoantibodies (TAAb. However, the independent contribution of SPB and TAAb expression data for identifying BC relative to a combinatorial SPB and TAAb approach has not been fully investigated. This study evaluates these contributions using a retrospective cohort of pre-biopsy serum samples with known clinical outcomes collected from a single site, thus minimizing potential site-to-site variation and enabling direct assessment of SPB and TAAb contributions to identify BC. All serum samples (n = 210 were collected prior to biopsy. These specimens were obtained from 18 participants with no evidence of breast disease (ND, 92 participants diagnosed with Benign Breast Disease (BBD and 100 participants diagnosed with BC, including DCIS. All BBD and BC diagnoses were based on pathology results from biopsy. Statistical models were developed to differentiate BC from non-BC (i.e., BBD and ND using expression data from SPB alone, TAAb alone, and a combination of SPB and TAAb. When SPB data was independently used for modeling, clinical sensitivity and specificity for detection of BC were 74.7% and 77.0%, respectively. When TAAb data was independently used, clinical sensitivity and specificity for detection of BC were 72.2% and 70.8%, respectively. When modeling integrated data from both SPB and TAAb, the clinical sensitivity and specificity for detection of BC improved to 81.0% and 78.8%, respectively. These data demonstrate the benefit of the integration of SPB and TAAb data and strongly support the further development of

A Novel Panel of Serum Biomarkers for MPM Diagnosis

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A. Bonotti

2017-01-01

Full Text Available Exposure to asbestos is the main cause of malignant pleural mesothelioma (MPM, a highly aggressive cancer of the pleura. Since the only tools for early detection are based on radiological tests, some authors focused on serum markers (i.e., mesothelin. The aim of this study was the evaluation of new serum biomarkers to be used individually or in combination, in order to improve the outcome of patients whose disease would be diagnosed at an earlier stage. Serum and plasma were available from 43 subjects previously exposed to asbestos and 27 MPM patients, all being epithelioid type. All the new markers found differentially expressed in MPM and healthy subjects, by proteomic and genomic approaches, have been validated in the serum by the use of specific ELISA. The combined approach, using tools of genomics and proteomics, is found to be highly innovative for this type of disease and led to the identification of new serum markers in the diagnosis of MPM. These results, if confirmed in a larger series, may have a strong impact in this area, because early detection of this cancer in people at high risk could significantly improve the course of the disease and the clinical approach to an individualized therapy.

Ibuprofen administration attenuates serum TNF-α levels, hepatic glutathione depletion, hepatic apoptosis and mouse mortality after Fas stimulation

International Nuclear Information System (INIS)

Cazanave, Sophie; Vadrot, Nathalie; Tinel, Marina; Berson, Alain; Letteron, Philippe; Larosche, Isabelle; Descatoire, Veronique; Feldmann, Gerard; Robin, Marie-Anne; Pessayre, Dominique

2008-01-01

Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-α (TNF-α), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 μg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-α. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-α secretion) and infliximab (trapping TNF-α) likewise attenuated the Jo2-mediated increase in TNF-α, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-α secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-α secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice

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.

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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

Serum cytokines, T lymphocyte subsets and STAT3 function in patients with herpes zoster as well as the intervention effect of mouse nerve growth factor

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Yun Xu

2016-07-01

Full Text Available Objective: To assess the levels of serum cytokines, T lymphocyte subsets and STAT3 in patients with herpes zoster as well as the intervention effect of mouse nerve growth factor. Methods: A total of 102 patients with herpes zoster were selected as observation group and received mouse nerve growth factor intervention, and 100 cases of normal people who received physical examination in our hospital during the same period as the healthy control group. The levels of serum Th1/Th2 cytokines, IgG subclass and complements and T lymphocyte subsets as well as STAT3 function of observation group before and after treatment and healthy control group were detected. Results: Serum IL-2 and γ-IFN levels of observation group after treatment were higher than those before treatment while IL-4, IL-5, IL-10 and TNF-α毩 levels were lower than those before treatment (P<0.05; serum IgG1, IgG3, IgG4, C3 and C4 values of observation group after treatment were higher than those before treatment while IgG2 value was lower than that before treatment (P<0.05; CD3, CD4 and CD4/CD8 levels of observation group after treatment were higher than those before treatment while CD8 level was lower than that before treatment (P<0.05; STAT3, p-STAT3 and JAK2 expression levels of observation group after treatment were lower than those before treatment (P<0.05. Conclusions: There are abnormal immune system and STAT3 signaling pathway function in patients with herpes zoster, and mouse nerve growth factor intervention can restore multisystem balance and accelerate disease rehabilitation, and has positive clinical significance.

Integration of cardiac proteome biology and medicine by a specialized knowledgebase.

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Zong, Nobel C; Li, Haomin; Li, Hua; Lam, Maggie P Y; Jimenez, Rafael C; Kim, Christina S; Deng, Ning; Kim, Allen K; Choi, Jeong Ho; Zelaya, Ivette; Liem, David; Meyer, David; Odeberg, Jacob; Fang, Caiyun; Lu, Hao-Jie; Xu, Tao; Weiss, James; Duan, Huilong; Uhlen, Mathias; Yates, John R; Apweiler, Rolf; Ge, Junbo; Hermjakob, Henning; Ping, Peipei

2013-10-12

Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo.

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Novoa-Herran, Susana; Umaña-Perez, Adriana; Canals, Francesc; Sanchez-Gomez, Myriam

2016-01-01

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line. The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test ( n  = 3, significance level 0.10, D > 0.642) and/or ANOVA ( n  = 3, p  depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells. This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that

Proteome-wide Adaptations of Mouse Skeletal Muscles during a Full Month in Space.

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Tascher, Georg; Brioche, Thomas; Maes, Pauline; Chopard, Angèle; O'Gorman, Donal; Gauquelin-Koch, Guillemette; Blanc, Stéphane; Bertile, Fabrice

2017-07-07

The safety of space flight is challenged by a severe loss of skeletal muscle mass, strength, and endurance that may compromise the health and performance of astronauts. The molecular mechanisms underpinning muscle atrophy and decreased performance have been studied mostly after short duration flights and are still not fully elucidated. By deciphering the muscle proteome changes elicited in mice after a full month aboard the BION-M1 biosatellite, we observed that the antigravity soleus incurred the greatest changes compared with locomotor muscles. Proteomics data notably suggested mitochondrial dysfunction, metabolic and fiber type switching toward glycolytic type II fibers, structural alterations, and calcium signaling-related defects to be the main causes for decreased muscle performance in flown mice. Alterations of the protein balance, mTOR pathway, myogenesis, and apoptosis were expected to contribute to muscle atrophy. Moreover, several signs reflecting alteration of telomere maintenance, oxidative stress, and insulin resistance were found as possible additional deleterious effects. Finally, 8 days of recovery post flight were not sufficient to restore completely flight-induced changes. Thus in-depth proteomics analysis unraveled the complex and multifactorial remodeling of skeletal muscle structure and function during long-term space flight, which should help define combined sets of countermeasures before, during, and after the flight.

Proteomics for discovery of candidate colorectal cancer biomarkers

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Álvarez-Chaver, Paula; Otero-Estévez, Olalla; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco J; Martínez-Zorzano, Vicenta S

2014-01-01

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers. PMID:24744574

Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13

KAUST Repository

Narasimhan, Kothandaraman

2013-02-01

Objectives: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. Methods: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Results: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Conclusions: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies. © 2013 John Wiley & Sons, Ltd.

Proteomic analysis of serum of workers occupationally exposed to arsenic, cadmium, and lead for biomarker research: A preliminary study

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Kossowska, Barbara, E-mail: barbara@immchem.am.wroc.pl [Department of Chemistry and Immunochemistry, Wroclaw Medical University, Bujwida 44a, 50-345 Wroclaw (Poland); Dudka, Ilona, E-mail: ilona.dudka@pwr.wroc.pl [Medicinal Chemistry and Microbiology Group, Department of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw (Poland); Bugla-Ploskonska, Gabriela, E-mail: gabriela.bugla-ploskonska@microb.uni.wroc.pl [Department of Microbiology, Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw (Poland); Szymanska-Chabowska, Anna, E-mail: aszyman@mp.pl [Department of Internal and Occupational Medicine, Wroclaw Medical University, Wybrzeze L. Pasteura 4, 50-367 Wroclaw (Poland); Doroszkiewicz, Wlodzimierz, E-mail: wlodzimierz.doroszkiewicz@microb.uni.wroc.pl [Department of Microbiology, Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw (Poland); Gancarz, Roman, E-mail: roman.gancarz@pwr.wroc.pl [Medicinal Chemistry and Microbiology Group, Department of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw (Poland); Andrzejak, Ryszard, E-mail: ryszard@chzaw.am.wroc.pl [Department of Internal and Occupational Medicine, Wroclaw Medical University, Wybrzeze L. Pasteura 4, 50-367 Wroclaw (Poland); Antonowicz-Juchniewicz, Jolanta, E-mail: jola@chzaw.am.wroc.pl [Department of Internal and Occupational Medicine, Wroclaw Medical University, Wybrzeze L. Pasteura 4, 50-367 Wroclaw (Poland)

2010-10-15

The main factor of environmental contamination is the presence of the heavy metals lead, cadmium, and arsenic. The aim of serum protein profile analysis of people chronically exposed to heavy metals is to find protein markers of early pathological changes. The study was conducted in a group of 389 healthy men working in copper foundry and 45 age-matched non-exposed healthy men. Toxicological test samples included whole blood, serum, and urine. Thirty-seven clinical parameters were measured. Based on the parameters values of the healthy volunteers, the centroid in 37-dimensional space was calculated. The individuals in the metal-exposed and control groups were ordered based on the Euclidean distance from the centroid defined by the first component according to Principal Component Analysis (PCA). Serum samples of two individuals, one from the control and one from the metal-exposed group, were chosen for proteomic analysis. In optimized conditions of two-dimensional gel electrophoresis (2-DE), two protein maps were obtained representing both groups. Twenty-eight corresponding protein spots from both protein maps were chosen and identified based on PDQuest analysis and the SWISS-2DPAGE database. From a panel of six proteins with differences in expression greater than a factor of two, three potential markers with the highest differences were selected: hemoglobin-spot 26 (pI 7.05, Mw 10.53), unidentified protein-spot 27 (pI 6.73, Mw 10.17), and unidentified protein-spot 25 (pI 5.75, Mw 12.07). Further studies are required to prove so far obtained results. Identified proteins could serve as potential markers of preclinical changes and could be in the future included in biomonitoring of people exposed to heavy metals.

Proteomic analysis of serum of workers occupationally exposed to arsenic, cadmium, and lead for biomarker research: A preliminary study

International Nuclear Information System (INIS)

Kossowska, Barbara; Dudka, Ilona; Bugla-Ploskonska, Gabriela; Szymanska-Chabowska, Anna; Doroszkiewicz, Wlodzimierz; Gancarz, Roman; Andrzejak, Ryszard; Antonowicz-Juchniewicz, Jolanta

2010-01-01

The main factor of environmental contamination is the presence of the heavy metals lead, cadmium, and arsenic. The aim of serum protein profile analysis of people chronically exposed to heavy metals is to find protein markers of early pathological changes. The study was conducted in a group of 389 healthy men working in copper foundry and 45 age-matched non-exposed healthy men. Toxicological test samples included whole blood, serum, and urine. Thirty-seven clinical parameters were measured. Based on the parameters values of the healthy volunteers, the centroid in 37-dimensional space was calculated. The individuals in the metal-exposed and control groups were ordered based on the Euclidean distance from the centroid defined by the first component according to Principal Component Analysis (PCA). Serum samples of two individuals, one from the control and one from the metal-exposed group, were chosen for proteomic analysis. In optimized conditions of two-dimensional gel electrophoresis (2-DE), two protein maps were obtained representing both groups. Twenty-eight corresponding protein spots from both protein maps were chosen and identified based on PDQuest analysis and the SWISS-2DPAGE database. From a panel of six proteins with differences in expression greater than a factor of two, three potential markers with the highest differences were selected: hemoglobin-spot 26 (pI 7.05, Mw 10.53), unidentified protein-spot 27 (pI 6.73, Mw 10.17), and unidentified protein-spot 25 (pI 5.75, Mw 12.07). Further studies are required to prove so far obtained results. Identified proteins could serve as potential markers of preclinical changes and could be in the future included in biomonitoring of people exposed to heavy metals.

Proteomic Analysis of Mouse Oocytes Reveals 28 Candidate Factors of the "Reprogrammome"

NARCIS (Netherlands)

Pfeiffer, M.J.; Siatkowski, M.; Paudel, Y.; Balbach, S.T.; Baeumer, N.; Crosetto, N.; Drexler, H.C.A.; Fuellen, G.; Boiani, M.

2011-01-01

The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date,

Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection.

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Li, S; Tseng, W C; Stolz, D B; Wu, S P; Watkins, S C; Huang, L

1999-04-01

Intravenous gene delivery via cationic lipidic vectors gives systemic gene expression particularly in the lung. In order to understand the mechanism of intravenous lipofection, a systematic study was performed to investigate the interactions of lipidic vectors with mouse serum emphasizing how serum affects the biophysical and biological properties of vectors of different lipid compositions. Results from this study showed that lipidic vectors underwent dynamic changes in their characteristics after exposure to serum. Addition of lipidic vectors into serum resulted in an immediate aggregation of vectors. Prolonged incubation of lipidic vectors with serum led to vector disintegration as shown in turbidity study, sucrose-gradient centrifugation analysis and fluorescence resonance energy transfer (FRET) study. Vector disintegration was associated with DNA release and degradation as shown in EtBr intercalation assay and DNA digestion study. Serum-induced disintegration of vectors is a general phenomenon for all cationic lipidic vectors tested in this study. Yet, vectors of different lipid compositions vary greatly in the rate of disintegration. There is an inverse correlation between the disintegration rate of lipidic vectors and their in vivo transfection efficiency. Vectors with a rapid rate of disintegration such as those containing dioleoyl-phosphatidylethanolamine (DOPE) poorly stayed in the lung and were barely active in transfecting cells. In contrast, cholesterol-containing vectors that had a rapid aggregation and a slow disintegration were highly efficient in transfecting cells in vivo. The results of this study explain why cationic lipidic vectors of different lipid compositions have a dramatic difference in their in vivo transfection efficiency. These results also suggest that the study of the interactions of lipidic vectors with serum may serve as a predictive model for the in vivo efficiency of a lipidic vector. Further study of the numerous interactions of

Murine colon proteome and characterization of the protein pathways

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Magdeldin Sameh

2012-08-01

Full Text Available Abstract Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR I and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9 in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2, Glutathione S-transferase (Gstp1 in prostate cancer, and Cell division control protein (Cdc42, Ras-related protein (Rac1,2 in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

A Comparative Proteome Profile of Female Mouse Gonads Suggests a Tight Link between the Electron Transport Chain and Meiosis Initiation.

Science.gov (United States)

Shen, Cong; Li, Mingrui; Zhang, Pan; Guo, Yueshuai; Zhang, Hao; Zheng, Bo; Teng, Hui; Zhou, Tao; Guo, Xuejiang; Huo, Ran

2018-01-01

Generation of haploid gametes by meiosis is a unique property of germ cells and is critical for sexual reproduction. Leaving mitosis and entering meiosis is a key step in germ cell development. Several inducers or intrinsic genes are known to be important for meiotic initiation, but the regulation of meiotic initiation, especially at the protein level, is still not well understood. We constructed a comparative proteome profile of female mouse fetal gonads at specific time points (11.5, 12.5, and 13.5 days post coitum), spanning a critical window for initiation of meiosis in female germ cells. We identified 3666 proteins, of which 473 were differentially expressed. Further bioinformatics analysis showed that these differentially expressed proteins were enriched in the mitochondria, especially in the electron transport chain and, notably, 9 proteins in electron transport chain Complex I were differentially expressed. We disrupted the mitochondrial electron transport chain function by adding the complex I inhibitor, rotenone to 11.5 days post coitum female gonads cultured in vitro. This treatment resulted in a decreased proportion of meiotic germ cells, as assessed by staining for histone γH2AX. Rotenone treatment also caused decreased ATP levels, increased reactive oxygen species levels and failure of the germ cells to undergo premeiotic DNA replication. These effects were partially rescued by adding Coenzyme Q10. Taken together, our results suggested that a functional electron transport chain is important for meiosis initiation. Our characterization of the quantitative proteome of female gonads provides an inventory of proteins, useful for understanding the mechanisms of meiosis initiation and female fertility. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic biomarkers predicting lymph node involvement in serum of cervical cancer patients. Limitations of SELDI-TOF MS

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Van Gorp Toon

2012-06-01

Full Text Available Abstract Background Lymph node status is not part of the staging system for cervical cancer, but provides important information for prognosis and treatment. We investigated whether lymph node status can be predicted with proteomic profiling. Material & methods Serum samples of 60 cervical cancer patients (FIGO I/II were obtained before primary treatment. Samples were run through a HPLC depletion column, eliminating the 14 most abundant proteins ubiquitously present in serum. Unbound fractions were concentrated with spin filters. Fractions were spotted onto CM10 and IMAC30 surfaces and analyzed with surface-enhanced laser desorption time of flight (SELDI-TOF mass spectrometry (MS. Unsupervised peak detection and peak clustering was performed using MASDA software. Leave-one-out (LOO validation for weighted Least Squares Support Vector Machines (LSSVM was used for prediction of lymph node involvement. Other outcomes were histological type, lymphvascular space involvement (LVSI and recurrent disease. Results LSSVM models were able to determine LN status with a LOO area under the receiver operating characteristics curve (AUC of 0.95, based on peaks with m/z values 2,698.9, 3,953.2, and 15,254.8. Furthermore, we were able to predict LVSI (AUC 0.81, to predict recurrence (AUC 0.92, and to differentiate between squamous carcinomas and adenocarcinomas (AUC 0.88, between squamous and adenosquamous carcinomas (AUC 0.85, and between adenocarcinomas and adenosquamous carcinomas (AUC 0.94. Conclusions Potential markers related with lymph node involvement were detected, and protein/peptide profiling support differentiation between various subtypes of cervical cancer. However, identification of the potential biomarkers was hampered by the technical limitations of SELDI-TOF MS.

Analytical methods for proteome data obtained from SDS-PAGE multi-dimensional separation and mass spectrometry

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Gun Wook Park

2010-03-01

Full Text Available For proteome analysis, various experimental protocols using mass spectrometry have been developed over thelast decade. The different protocols have differing performances and degrees of accuracy. Furthermore, the “best”protocol for a proteomic analysis of a sample depends on the purpose of the analysis, especially in connection withdisease proteomics, including biomarker discovery and therapeutics analyses of human serum or plasma. Theprotein complexity and the wide dynamic range of blood samples require high-dimensional separation technology.In this article, we review proteome analysis protocols in which both Sodium Dodecyl Sulfate-Polyacryl Amide GelElectrophoresis(SDS-PAGE and liquid chromatography are used for peptide and protein separations. Multidimensionalseparation technology supplies a high-quality dataset of tandem mass spectra and reveals signals fromlow-abundance proteins, although it can be time-consuming and laborious work. We survey shotgun proteomicsprotocols using SDS-PAGE and liquid chromatography and introduce bioinformatics tools for the analysis ofproteomics data. We also review efforts toward the biological interpretation of the proteome.

Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

Science.gov (United States)

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer’s disease

International Nuclear Information System (INIS)

Chang, Seong-Hun; Jung, In-Soo; Han, Gi-Yeon; Kim, Nam-Hee; Kim, Hyun-Jung; Kim, Chan-Wha

2013-01-01

Highlights: ► A transgenic mouse model expressing NSE-htau23 was used. ► 2D-gel electrophoresis to analyze the cortex proteins of transgenic mice was used. ► Differentially expressed spots in different stages of AD were identified. ► GSTP1 and CAII were downregulated with the progression of AD. ► SCRN1 and ATP6VE1 were up regulated and down regulated differentially. -- Abstract: Alzheimer’s disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

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Hsu Kimberly K

2005-06-01

Full Text Available Abstract Background The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. Results After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine, showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyldimethylammonio]-1-propanesulfonate. Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. Conclusion This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

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Vivek Phani Varma

Full Text Available Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO and fetal bovine serum (FBS is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis ocular fluid (BuOF to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4, mouse embryonic stem (mES cells, and primary cells, such as mouse embryonic fibroblast (MEF cells, human peripheral blood mononuclear cells (hPBMCs, and mouse bone marrow cells (mBMCs, were cryopreserved in cryomedia containing 10% DMSO (D10 with 20% FBS (D10S20 or D10 with 20% BuOF (D10O20. For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types

Proteomics Core

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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...

Quantitative Evaluation of Serum Proteins Uncovers a Protein Signature Related to Maturity-Onset Diabetes of the Young (MODY).

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Tuerxunyiming, Muhadasi; Xian, Feng; Zi, Jin; Yimamu, Yilihamujiang; Abuduwayite, Reshalaiti; Ren, Yan; Li, Qidan; Abudula, Abulizi; Liu, SiQi; Mohemaiti, Patamu

2018-01-05

Maturity-onset diabetes of the young (MODY) is an inherited monogenic type of diabetes. Genetic mutations in MODY often cause nonsynonymous changes that directly lead to the functional distortion of proteins and the pathological consequences. Herein, we proposed that the inherited mutations found in a MODY family could cause a disturbance of protein abundance, specifically in serum. The serum samples were collected from a Uyghur MODY family through three generations, and the serum proteins after depletion treatment were examined by quantitative proteomics to characterize the MODY-related serum proteins followed by verification using target quantification of proteomics. A total of 32 serum proteins were preliminarily identified as the MODY-related. Further verification test toward the individual samples demonstrated the 12 candidates with the significantly different abundance in the MODY patients. A comparison of the 12 proteins among the sera of type 1 diabetes, type 2 diabetes, MODY, and healthy subjects was conducted and revealed a protein signature related with MODY composed of the serum proteins such as SERPINA7, APOC4, LPA, C6, and F5.

Nutritional Proteomics: Investigating molecular mechanisms underlying the health beneficial effect of functional foods

Directory of Open Access Journals (Sweden)

Yusuke Kawashima

2013-07-01

Full Text Available ABSTRACTObjective: We introduce a new technical and conceptual term “nutritional proteomics” by identifying and quantifying the proteins and their changes in a certain organ or tissue dependent on the food intake by utilizing a mass spectrometry-based proteomics technique.Purpose: Food intake is essentially important for every life on earth to sustain the physical as well as mental functions. The outcome of food intake will be manifested in the health state and its dysfunction. The molecular information about the protein expression change caused by diets will assist us to understand the significance of functional foods. We wish to develop nutritional proteomics to promote a new area in functional food studies for a better understanding of the role of functional foods in health and disease.Methods: We chose two classes of food ingredients to show the feasibility of nutritional proteomics, omega-3 polyunsaturated fatty acids and omega-6 polyunsaturated fatty acids both of which are involved in the inflammation/anti-inflammation axis. Each class of the polyunsaturated fatty acids was mixed in mouse chow respectively. The liver tissue of mice fed with omega-3 diet or omega-3 diet was analyzed by the state-of-the-art shotgun proteomics using nano-HPLC-ESI-MS/MS. The data were analyzed by the number of differentially expressed proteins that were guaranteed by 1% false discovery rate for protein identification and by the statistical significance of variance evaluated by p-value in two-tailed distribution analysis better than 0.05 (n=4. The differential pattern of protein expression was characterized with Gene Ontology designation.Results: The data analysis of the shotgun nutritional proteomics identified 2,810 proteins that are validated with 1% FDR. Among these 2,810 proteins, 125 were characterized with statistical significance of variance (p<0.05; n=4 between the omega-3 diet and the omega-6 diet by twotailed distribution analysis. The results

The effect of microbial colonization on the host proteome varies by gastrointestinal location.

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Lichtman, Joshua S; Alsentzer, Emily; Jaffe, Mia; Sprockett, Daniel; Masutani, Evan; Ikwa, Elvis; Fragiadakis, Gabriela K; Clifford, David; Huang, Bevan Emma; Sonnenburg, Justin L; Huang, Kerwyn Casey; Elias, Joshua E

2016-05-01

Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.

Rhythmic Degradation Explains and Unifies Circadian Transcriptome and Proteome Data

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Sarah Lück

2014-10-01

Full Text Available The rich mammalian cellular circadian output affects thousands of genes in many cell types and has been the subject of genome-wide transcriptome and proteome studies. The results have been enigmatic because transcript peak abundances do not always follow the peaks of gene-expression activity in time. We posited that circadian degradation of mRNAs and proteins plays a pivotal role in setting their peak times. To establish guiding principles, we derived a theoretical framework that fully describes the amplitudes and phases of biomolecules with circadian half-lives. We were able to explain the circadian transcriptome and proteome studies with the same unifying theory, including cases in which transcripts or proteins appeared before the onset of increased production rates. Furthermore, we estimate that 30% of the circadian transcripts in mouse liver and Drosophila heads are affected by rhythmic posttranscriptional regulation.

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

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Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias

2012-03-01

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

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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.

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer's disease

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Chang, Seong-Hun; Jung, In-Soo; Han, Gi-Yeon; Kim, Nam-Hee; Kim, Hyun-Jung [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Chan-Wha, E-mail: cwkim@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

2013-01-11

Highlights: Black-Right-Pointing-Pointer A transgenic mouse model expressing NSE-htau23 was used. Black-Right-Pointing-Pointer 2D-gel electrophoresis to analyze the cortex proteins of transgenic mice was used. Black-Right-Pointing-Pointer Differentially expressed spots in different stages of AD were identified. Black-Right-Pointing-Pointer GSTP1 and CAII were downregulated with the progression of AD. Black-Right-Pointing-Pointer SCRN1 and ATP6VE1 were up regulated and down regulated differentially. -- Abstract: Alzheimer's disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The

Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation

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Pedersen, Brian A; Yazdi, Puya G; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Wang, Ping H

2015-01-01

Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart. PMID:26610654

Clinical proteomics

DEFF Research Database (Denmark)

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...

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

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Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B; Hettinga, Kasper

2016-09-16

To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the

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

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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.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

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Peter A. Bell

2013-06-01

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Candidate proteomic biomarkers for non-alcoholic fatty liver disease (steatosis and non-alcoholic steatohepatitis) discovered with mass-spectrometry: a systematic review.

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Lădaru, Anca; Bălănescu, Paul; Stan, Mihaela; Codreanu, Ioana; Anca, Ioana Alina

2016-01-01

Non-alcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver which is accompanied by a series of metabolic deregulations. There are sustained research efforts focusing upon biomarker discovery for NAFLD diagnosis and its prognosis in order investigate and follow-up patients as minimally invasive as possible. The objective of this study is to critically review proteomic studies that used mass spectrometry techniques and summarize relevant proteomic NAFLD candidate biomarkers. Medline and Embase databases were searched from inception to December 2014. A final number of 22 records were included that identified 251 candidate proteomic biomarkers. Thirty-three biomarkers were confirmed - 14 were found in liver samples, 21 in serum samples, and two from both serum and liver samples. Some of the biomarkers identified have already been extensively studied regarding their diagnostic and prognostic capacity. However, there are also more potential biomarkers that still need to be addressed in future studies.

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

International Nuclear Information System (INIS)

Roblin, R.O.; Bell, T.E.; Young, P.L.

1978-01-01

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radiommunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply. (Auth.)

Aptamer-based multiplexed proteomic technology for biomarker discovery.

Directory of Open Access Journals (Sweden)

Larry Gold

2010-12-01

Full Text Available The interrogation of proteomes ("proteomics" in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma. Our current assay measures 813 proteins with low limits of detection (1 pM median, 7 logs of overall dynamic range (~100 fM-1 µM, and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD. We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

Science.gov (United States)

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N; Carter, Jeff; Dalby, Andrew B; Eaton, Bruce E; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R; Kim, Nancy; Koch, Tad H; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D; Vrkljan, Mike; Walker, Jeffrey J; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K; Wolfson, Alexey; Wolk, Steven K; Zhang, Chi; Zichi, Dom

2010-12-07

The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Identification and proteomic analysis of osteoblast-derived exosomes

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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.

Biochemical Markers of Brain Injury: An Integrated Proteomics-Based Approach

Science.gov (United States)

2006-02-01

some variability between protein assay measurements. Using densitometric analysis after 1D-PAGE, microfiltration alone showed an 11 ( 5% sample reduction...KOPETSCH, O., WOSZCZYK, A., et al. (2003). Serum S-100B protein as a molecular marker in severe trau- matic brain injury. Restor . Neurol. Neurosci. 21...proteomics. Implications in the search for preventive initiatives to slow the clinical progression of Alzheimer’s disease dementia. Restor . Neurol. Neurosci

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

Science.gov (United States)

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

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

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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

Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

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Patricia eNAGNAN-LE MEILLOUR

2014-12-01

Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

The Succinated Proteome

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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-enzymatic post-translational modification of proteins by proteomics approaches.

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

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Kai P. Law

2015-05-01

Full Text Available Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered.

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

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Law, Kai P.; Han, Ting-Li; Tong, Chao; Baker, Philip N.

2015-01-01

Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered. PMID:26006232

'Gate effect' in templated polyacrylamide membranes influences the electrotransport of proteins and finds applications in proteome analysis.

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Bossi, Alessandra; Andreoli, Matteo; Bonini, Francesca; Piletsky, Sergey

2007-09-01

Templating is an effective way for the structural modifications of a material and hence for altering its functional properties. Here protein imprinting was exploited to alter polymeric polyacrylamide (PAA) membranes. The sieving properties and selection abilities of the material formed were evaluated by studying the electrically driven transport of various proteins across templated PAA membranes. The sieving properties correlated with the templating process and depended on the quantity of template used during the polymerisation. For 1 mg/mL protein-templated membranes a 'gate effect' was shown, which induced a preferential migration of the template and of similar-size proteins. Such template preferential electrotransport was exploited for the selective removal of certain proteins in biological fluids prior to proteome analysis (depletion of albumin from human serum); the efficiency of the removal was demonstrated by analysing the serum proteome by two-dimensional electrophoresis experiments.

GeLC-MS: A Sample Preparation Method for Proteomics Analysis of Minimal Amount of Tissue.

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Makridakis, Manousos; Vlahou, Antonia

2017-10-10

Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology. Despite the significant advances in the mass spectrometry instrumentation, tissue proteomics still faces several challenges mainly due to increased sample complexity and heterogeneity. However, the most prominent challenge is attributed to the invasive procedure of tissue sampling which restricts the availability of fresh frozen tissue to minimal amounts and limited number of samples. Application of GeLC-MS sample preparation protocol for tissue proteomics analysis can greatly facilitate making up for these difficulties. In this chapter, a step by step guide for the proteomics analysis of minute amounts of tissue samples using the GeLC-MS sample preparation protocol, as applied by our group in the analysis of multiple different types of tissues (vessels, kidney, bladder, prostate, heart) is provided.

Serum Metabonomics of Mild Acute Pancreatitis.

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Xu, Hongmin; Zhang, Lei; Kang, Huan; Zhang, Jiandong; Liu, Jie; Liu, Shuye

2016-11-01

Mild acute pancreatitis (MAP) is a common acute abdominal disease, and exhibits rising incidence in recent decades. As an important component of systemic biology, metabonomics is a new discipline developed following genomics and proteomics. In this study, the objective was to analyze the serum metabonomics of patients with MAP, aiming to screen metabolic markers with potential diagnostic values. An analysis platform with ultra performance liquid chromatography-high-resolution mass spectrometry was used to screen the difference metabolites related to MAP diagnosis and disease course monitoring. A total of 432 endogenous metabolites were screened out from 122 serum samples, and 49 difference metabolites were verified, among which 12 difference metabolites were identified by nonparametric test. After material identification, eight metabolites exhibited reliable results, and their levels in MAP serum were higher than those in healthy serum. Four metabolites exhibited gradual downward trend with treatment process going on, and the differences were statistically significant (P Metabonomic analysis has revealed eight metabolites with potential diagnostic values toward MAP, among which four metabolites can be used to monitor the disease course. © 2016 Wiley Periodicals, Inc.

Characterization of the porcine synovial fluid proteome and a comparison to the plasma proteome

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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.

ProteomicsDB.

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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.

Development of gel-filter method for high enrichment of low-molecular weight proteins from serum.

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Lingsheng Chen

Full Text Available The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ≤ 30kDa difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2 from 10 μL serum. Among them, 559 (n = 2 proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.

Comparative proteomics in alkaptonuria provides insights into inflammation and oxidative stress.

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Braconi, Daniela; Bernardini, Giulia; Paffetti, Alessandro; Millucci, Lia; Geminiani, Michela; Laschi, Marcella; Frediani, Bruno; Marzocchi, Barbara; Santucci, Annalisa

2016-12-01

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism associated with a defective catabolism of phenylalanine and tyrosine leading to increased systemic levels of homogentisic acid (HGA). Excess HGA is partly excreted in the urine, partly accumulated within the body and deposited onto connective tissues under the form of an ochronotic pigment, leading to a range of clinical manifestations. No clear genotype/phenotype correlation was found in AKU, and today there is the urgent need to identify biomarkers able to monitor AKU progression and evaluate response to treatment. With this aim, we provided the first proteomic study on serum and plasma samples from alkaptonuric individuals showing pathological SAA, CRP and Advanced Oxidation Protein Products (AOPP) levels. Interesting similarities with proteomic studies on other rheumatic diseases were highlighted together with proteome alterations supporting the existence of oxidative stress and inflammation in AKU. Potential candidate biomarkers to assess disease severity, monitor disease progression and evaluate response to treatment were identified as well. Copyright © 2016 Elsevier Ltd. All rights reserved.

Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.

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Riebeling, Christian; Schlechter, Katharina; Buesen, Roland; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-06-01

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST. Copyright © 2011 Elsevier Ltd. All rights reserved.

Building ProteomeTools based on a complete synthetic human proteome

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Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard

2018-01-01

The ProteomeTools project builds molecular and digital tools from the human proteome to facilitate biomedical and life science research. Here, we report the generation and multimodal LC-MS/MS analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products and exemplify the utility of this data. The resource will be extended to >1 million peptides and all data will be shared with the community via ProteomicsDB and proteomeXchange. PMID:28135259

Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27

DEFF Research Database (Denmark)

Soler, Laura; Miller, Ingrid; Hummel, Karin

2016-01-01

to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation...

Proteome analysis of the hypercholestrolemic rat, RICO

International Nuclear Information System (INIS)

Cho, S.Y.; Park, K.-S.; Paik, Y.-K.; Seong, J.-K.

2001-01-01

In an attempt to develop novel markers for hypercholesterolemia, hepatic tissues and serum prepared from hypeicholesterolemic rat (i e RICO) were analyzed by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-ToF). Results were compared to those of paired inbreed rat (WKY). Comparative analysis of the respective spot patterns in 2DE revealed that the numbers of differential expression proteins were identified in serum and liver tissues of RICO. Some of the representative proteins annotated in 2DE were apolipoprotein family and numerous lipid metabolism related proteins. Especially, we found that protein disulfide isomerase subunits (ER-60) in 2DE have differential post-translational modification pattern by MALDI-ToF analysis. Our results suggest that the proteomic analysis of these proteins might be a novel approach to identify the molecular events in detail during lipid disorder such atherosclerosis

Overlap of proteomics biomarkers between women with pre-eclampsia and PCOS: a systematic review and biomarker database integration.

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Khan, Gulafshana Hafeez; Galazis, Nicolas; Docheva, Nikolina; Layfield, Robert; Atiomo, William

2015-01-01

Do any proteomic biomarkers previously identified for pre-eclampsia (PE) overlap with those identified in women with polycystic ovary syndrome (PCOS). Five previously identified proteomic biomarkers were found to be common in women with PE and PCOS when compared with controls. Various studies have indicated an association between PCOS and PE; however, the pathophysiological mechanisms supporting this association are not known. A systematic review and update of our PCOS proteomic biomarker database was performed, along with a parallel review of PE biomarkers. The study included papers from 1980 to December 2013. In all the studies analysed, there were a total of 1423 patients and controls. The number of proteomic biomarkers that were catalogued for PE was 192. Five proteomic biomarkers were shown to be differentially expressed in women with PE and PCOS when compared with controls: transferrin, fibrinogen α, β and γ chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. In PE, the biomarkers were identified in serum, plasma and placenta and in PCOS, the biomarkers were identified in serum, follicular fluid, and ovarian and omental biopsies. The techniques employed to detect proteomics have limited ability in identifying proteins that are of low abundance, some of which may have a diagnostic potential. The sample sizes and number of biomarkers identified from these studies do not exclude the risk of false positives, a limitation of all biomarker studies. The biomarkers common to PE and PCOS were identified from proteomic analyses of different tissues. This data amalgamation of the proteomic studies in PE and in PCOS, for the first time, discovered a panel of five biomarkers for PE which are common to women with PCOS, including transferrin, fibrinogen α, β and γ chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. If validated, these biomarkers could provide a useful framework for the knowledge infrastructure in this area. To accomplish this goal, a

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

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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.

Effects of chronic high stocking density on liver proteome of rainbow trout (Oncorhynchus mykiss).

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Naderi, Mahdi; Keyvanshokooh, Saeed; Salati, Amir Parviz; Ghaedi, Alireza

2017-10-01

The main aim of the present study was to assess the effects of chronic high stocking density on liver proteome of rainbow trout. Rainbow trout juveniles (42.6 ± 2.3 g average body weight) were randomly distributed into six tanks at two stocking densities (low stocking density (LD) = 20 kg m -3 and high stocking density (HD) = 80 kg m -3 ). Both treatments were performed in triplicate tanks for a period of 60 days. High stocking density caused a reduction in the growth performance compared with LD fish. Lysozyme activity increased with stocking density, while serum complement activity presented the opposite pattern. Serum cortisol and total protein levels did not show significant differences (P > 0.05) between experimental groups. The fish reared at high stocking density showed significantly lower osmolality and globulin values but higher albumin level. The HD group had significantly higher activities of catalase, glutathione peroxidase and superoxide dismutase, and malondialdehyde content in the liver when compared to the LD group. Comparative proteomics was used to determine the proteomic responses in livers of rainbow trout reared at high stocking density for 60 days. Out of nine protein spots showing altered abundance (>1.5-folds, P < 0.05), eight spots were successfully identified. Two proteins including apolipoprotein A-I-2 precursor and mitochondrial stress-70 protein were found to increase in HD group. The spots found to decrease in the HD group were identified as follows: 2-peptidylprolyl isomerase A, two isoforms of glyceraldehydes-3-phosphate dehydrogenase, an unnamed protein product similar to fructose-bisphosphate aldolase, 78 kDa glucose-regulated protein, and serum albumin 1 protein.

A microfluorometric assay for immunoglobulin class and subclass levels in murine serum

NARCIS (Netherlands)

Haaijman, J.J.; Brinkhof, J.

1977-01-01

The IgG fractions of rabbit antisera specific for the Fc part of mouse IgA, IgM, and the four subclasses of IgG (1, 2a, 2b, and 3) were coupled covalently to Sepharose beads by the cyanogen bromide method. The beads were then incubated with different dilutions of mouse serum. The mouse

Serum β-amyloid peptide levels spike in the early stage of Alzheimer-like plaque pathology in an APP/PS1 double transgenic mouse model.

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He, Jue; Qiao, Jin-Ping; Zhu, Shenghua; Xue, Mengzhou; Chen, Wenwu; Wang, Xinchun; Tempier, Adrien; Huang, Qingjun; Kong, Jiming; Li, Xin-Min

2013-11-01

Serum levels of β-amyloid (Aβ) peptides may represent an early biomarker in the diagnosis of Alzheimer's disease (AD). In the present study, we investigated the temporal kinetic changes in the levels of serum Aβ 1-42 and 40 in an amyloid precursor protein (APP)/presenilin (PS)1 double transgenic mouse model of AD. Serum Aβ peptide levels in 2-, 3-, 6-, 9- and 18-month old, and liver Aβ 1-40 level in 6-month old mice were measured using enzyme-linked immunosorbent assay (ELISA) kits. Results revealed that serum Aβ levels peaked in 3-month old transgenic mice, and the Aβ level in non-transgenic and transgenic mice is comparable in liver. Compared to the 6-month old transgenic mice, Congo red staining showed that the 3-month old transgenic mice had minimum brain Aβ plaques, corresponding to the early stage of Alzheimer-like plaque pathology, and confocal microscope images showed that the deposition of Aβ in their cerebral vessels was minimal. Furthermore, results of the water maze test, showed that memory was normal for the 3- month old transgenic mice when compared to age-matched non-transgenic mice. These results suggest that serum Aβ peptide levels may be peaked during the early stage of AD. Monitoring serum Aβ peptide levels in the potential AD population may provide an early diagnosis of AD prior to the appearance of clinical symptoms.

Morbility, clinical data and proteomic analysis of IUGR and AGA newborns at different gestational ages

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M.D. Ruiz-González

2016-12-01

Full Text Available The data are related to the proteomic analysis of 43 newborns with intrauterine growth retardation (IUGR and 45 newborns with appropriate weight for gestational age (AGA carried out by separation via 2DE and analyzed by MS–TOF/TOF. All newborns were separated into three gestational age groups, "Very Preterm" 29–32 weeks, "Moderate Preterm" 33–36 weeks, and, "Term" ≥37weeks. From each newborn, blood was drawn three times from birth to 1 month life. High-abundant serum proteins were depleted, and the minority ones were separated by 2DE and analyzed for significant expression differences. The data reflect analytic and clinic variables analyzed globally and categorized by gestational age in relation to IUGR and the optimization of conditions for 2-DE separation. The data from this study are related to the research article entitled "Alterations of Protein Expression in Serum of Infants with Intrauterine Growth Restriction and Different Gestational Ages" (M.D. Ruis-González, M.D. Cañete, J.L. Gómez-Chaparro, N. Abril, R. Cañete, J. López-Barea, 2015 [1]. The present dataset of serum IUGR newborn proteome can be used as a reference for any study involving intrauterine growth restriction during the first month of life.

Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection.

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Sengupta, Nabonita; Ghosh, Sourish; Vasaikar, Suhas V; Gomes, James; Basu, Anirban

2014-01-01

In this study we have reported the in vivo proteomic changes during Japanese Encephalitis Virus (JEV) infection in combination with in vitro studies which will help in the comprehensive characterization of the modifications in the host metabolism in response to JEV infection. We performed a 2-DE based quantitative proteomic study of JEV-infected mouse brain as well as mouse neuroblastoma (Neuro2a) cells to analyze the host response to this lethal virus. 56 host proteins were found to be differentially expressed post JEV infection (defined as exhibiting ≥ 1.5-fold change in protein abundance upon JEV infection). Bioinformatics analyses were used to generate JEV-regulated host response networks which reported that the identified proteins were found to be associated with various cellular processes ranging from intracellular protein transport, cellular metabolism and ER stress associated unfolded protein response. JEV was found to invade the host protein folding machinery to sustain its survival and replication inside the host thereby generating a vigorous unfolded protein response, subsequently triggering a number of pathways responsible for the JEV associated pathologies. The results were also validated using a human cell line to correlate them to the human response to JEV. The present investigation is the first report on JEV-host interactome in in vivo model and will be of potential interest for future antiviral research in this field.

Proteomics dataset

DEFF Research Database (Denmark)

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....

Mouse allergen-specific immunoglobulin G4 and risk of mouse skin test sensitivity

NARCIS (Netherlands)

Matsui, E. C.; Diette, G. B.; Krop, E. J. M.; Aalberse, R. C.; Smith, A. L.; Eggleston, P. A.

2006-01-01

High serum levels of cat-specific IgG and IgG4 are associated with protection against allergic sensitization to cat, but whether this association applies to other animal allergens remains unclear. To determine if high levels of mouse-specific IgG and IgG4 are associated with a decreased risk of

Proteomics research in India: an update.

Science.gov (United States)

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.

Comparative proteomic analysis of the molecular responses of mouse macrophages to titanium dioxide and copper oxide nanoparticles unravels some toxic mechanisms for copper oxide nanoparticles in macrophages.

Directory of Open Access Journals (Sweden)

Sarah Triboulet

Full Text Available Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide or of their biocidal properties (copper oxide, increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

Comparative proteomic analysis of the molecular responses of mouse macrophages to titanium dioxide and copper oxide nanoparticles unravels some toxic mechanisms for copper oxide nanoparticles in macrophages.

Science.gov (United States)

Triboulet, Sarah; Aude-Garcia, Catherine; Armand, Lucie; Collin-Faure, Véronique; Chevallet, Mireille; Diemer, Hélène; Gerdil, Adèle; Proamer, Fabienne; Strub, Jean-Marc; Habert, Aurélie; Herlin, Nathalie; Van Dorsselaer, Alain; Carrière, Marie; Rabilloud, Thierry

2015-01-01

Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses.

Directory of Open Access Journals (Sweden)

Lei Wang

Full Text Available Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days. Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

Effects of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse cumulus cell-oocyte complex.

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Camaioni, A; Hascall, V C; Yanagishita, M; Salustri, A

1993-09-25

Compact cumulus cell-oocyte complexes (COCs) isolated from preovulatory mouse follicles undergo expansion in vitro when high levels of hyaluronic acid (HA) are synthesized and organized into an extracellular matrix. We studied the effects of fetal bovine serum (FBS) and of exogenous HA and HA-oligomers on the expansion process. Maximum retention of HA in the COC matrix, and hence complete COC expansion, occurs when 1% FBS is continuously present during the first 18 h of culture. Irrespective of the culture time, HA synthesized when serum is absent is primarily in the medium, whereas HA synthesized when serum is present is primarily in the cell matrix. These findings support the hypothesis that the serum factor, identified as an inter-alpha-trypsin inhibitor by Chen et al. (Chen, L., Mao, S. J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), is a structural component of the matrix. Addition of exogenous HA or of HA oligomers of decasaccharide size (GlcUA-GlcNAc)5 or larger effectively displaces endogenously synthesized HA from the matrix into the medium, thereby preventing COC expansion. Addition of exogenous chondroitin sulfate affects neither matrix organization nor COC expansion, thus indicating specificity of the binding of some structural component(s) to HA. Fully expanded COCs disassemble when cultured longer than 18 h, a process which occurs also in vivo and which correlates with loss of oocyte fertilizability both in vivo and in vitro. This process involves release of macromolecular HA from the matrix into the medium, with loss of 50% of the HA in the first 8 h of incubation after full expansion. The release is not facilitated when HA oligomers, long enough to prevent matrix formation, are added to the culture medium after the COCs are fully expanded. This suggests that cooperative binding to HA of either the serum factor, an endogenously synthesized factor(s), or both is required to stabilize the fully expanded COC matrix.

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.

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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

Proteome analysis of body fluids for amyotrophic lateral sclerosis biomarker discovery.

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Krüger, Thomas; Lautenschläger, Janin; Grosskreutz, Julian; Rhode, Heidrun

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of motor neurons leading to death of the patients, mostly within 2-5 years after disease onset. The pathomechanism of motor neuron degeneration is only partially understood and therapeutic strategies based on mechanistic insights are largely ineffective. The discovery of reliable biomarkers of disease diagnosis and progression is the sine qua non of both the revelation of insights into the ALS pathomechanism and the assessment of treatment efficacies. Proteomic approaches are an important pillar in ALS biomarker discovery. Cerebrospinal fluid is the most promising body fluid for differential proteome analyses, followed by blood (serum, plasma), and even urine and saliva. The present study provides an overview about reported peptide/protein biomarker candidates that showed significantly altered levels in certain body fluids of ALS patients. These findings have to be discussed according to proposed pathomechanisms to identify modifiers of disease progression and to pave the way for the development of potential therapeutic strategies. Furthermore, limitations and advantages of proteomic approaches for ALS biomarker discovery in different body fluids and reliable validation of biomarker candidates have been addressed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The core proteome and pan proteome of Salmonella Paratyphi A epidemic strains.

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Li Zhang

Full Text Available Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

The Francisella tularensis proteome and its recognition by antibodies

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Sara L.N. Kilmury

2011-01-01

Full Text Available Francisella tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. The extreme virulence of the pathogen in humans, combined with the low infectious dose and the ease of dissemination by aerosol have led to concerns about its abuse as a bioweapon. Until recently, nothing was known about the virulence mechanisms and even now, there is still a relatively poor understanding of pathogen virulence. Completion of increasing numbers of Francisella genome sequences, combined with comparative genomics and proteomics studies, are contributing to the knowledge in this area. Tularemia may be treated with antibiotics, but there is currently no licensed vaccine. An attenuated strain, the Live Vaccine Strain (LVS has been used to vaccinate military and at risk laboratory personnel, but safety concerns mean that it is unlikely to be licensed by the FDA for general use. Little is known about the protective immunity induced by vaccination with LVS, in humans or animals models. Immunoproteomics studies with sera from infected humans or vaccinated mouse strains, are being used in gel based or proteome microarray approaches to give insight into the humoral immune response. In addition, these data have the potential to be exploited in the identification of new diagnostic or protective antigens, the design of next generation live vaccine strains, and the development of subunit vaccines. Herein, we briefly review the current knowledge from Francisella comparative proteomics studies and then focus upon the findings from immunoproteomics approaches.

Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

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Han, Dohyun; Jin, Jonghwa; Woo, Jongmin; Min, Hophil; Kim, Youngsoo

2014-07-01

Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole-cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte-neuron communication. To build a reference proteome, we established a C8-D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two-step digestion, filter-aided sample preparation, StageTip-based high pH fractionation, and high-resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High-confidence whole-cell proteomes and secretomes are valuable resources in studying astrocyte function by label-free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 (http://proteomecentral.proteomexchange.org/dataset/PXD000501). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Power of Proteomics in Linking Oxidative Stress and Female Infertility

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Gupta, Sajal; Sharma, Rakesh; Agarwal, Ashok

2014-01-01

Endometriosis, PCOS, and unexplained infertility are currently the most common diseases rendering large numbers of women infertile worldwide. Oxidative stress, due to its deleterious effects on proteins and nucleic acids, is postulated to be the one of the important mechanistic pathways in differential expression of proteins and in these diseases. The emerging field of proteomics has allowed identification of proteins involved in cell cycle, as antioxidants, extracellular matrix (ECM), cytoskeleton, and their linkage to oxidative stress in female infertility related diseases. The aim of this paper is to assess the association of oxidative stress and protein expression in the reproductive microenvironments such as endometrial fluid, peritoneal fluid, and follicular fluid, as well as reproductive tissues and serum. The review also highlights the literature that proposes the use of the fertility related proteins as potential biomarkers for noninvasive and early diagnosis of the aforementioned diseases rather than utilizing the more invasive methods used currently. The review will highlight the power of proteomic profiles identified in infertility related disease conditions and their linkage with underlying oxidative stress. The power of proteomics will be reviewed with regard to eliciting molecular mechanisms for early detection and management of these infertility related conditions. PMID:24900998

Power of Proteomics in Linking Oxidative Stress and Female Infertility

Directory of Open Access Journals (Sweden)

Sajal Gupta

2014-01-01

Full Text Available Endometriosis, PCOS, and unexplained infertility are currently the most common diseases rendering large numbers of women infertile worldwide. Oxidative stress, due to its deleterious effects on proteins and nucleic acids, is postulated to be the one of the important mechanistic pathways in differential expression of proteins and in these diseases. The emerging field of proteomics has allowed identification of proteins involved in cell cycle, as antioxidants, extracellular matrix (ECM, cytoskeleton, and their linkage to oxidative stress in female infertility related diseases. The aim of this paper is to assess the association of oxidative stress and protein expression in the reproductive microenvironments such as endometrial fluid, peritoneal fluid, and follicular fluid, as well as reproductive tissues and serum. The review also highlights the literature that proposes the use of the fertility related proteins as potential biomarkers for noninvasive and early diagnosis of the aforementioned diseases rather than utilizing the more invasive methods used currently. The review will highlight the power of proteomic profiles identified in infertility related disease conditions and their linkage with underlying oxidative stress. The power of proteomics will be reviewed with regard to eliciting molecular mechanisms for early detection and management of these infertility related conditions.

[Proteomics and transfusion medicine].

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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.

Efficacy of IgG and F(ab′)2 Antivenoms to Neutralize Snake Venom-induced Local Tissue Damage as Assessed by the Proteomic Analysis of Wound Exudate

OpenAIRE

Rucavado, Alexandra; Escalante Muñoz, Teresa; Shannon, John D.; Ayala Castro, Carla N.; Villalta, Mauren; Gutiérrez, José María; Fox, Jay W.

2012-01-01

2082-01 Embargo por política editorial Proteomic analysis of wound exudates represents a valuable tool to investigate tissue pathology and to assess the therapeutic success of various interventions. In this study, the ability of horse-derived IgG and F(ab0)2 antivenoms to neutralize local pathological effects induced by the venom of the snake Bothrops asper in mouse muscle was investigated by the proteomic analysis of exudates collected in the vicinity of affected tissue. In experiments...

CKD273, a new proteomics classifier assessing CKD and its prognosis.

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Ángel Argilés

Full Text Available National Kidney Foundation CKD staging has allowed uniformity in studies on CKD. However, early diagnosis and predicting progression to end stage renal disease are yet to be improved. Seventy six patients with different levels of CKD, including outpatients and dialysed patients were studied for transcriptome, metabolome and proteome description. High resolution urinary proteome analysis was blindly performed in the 53 non-anuric out of the 76 CKD patients. In addition to routine clinical parameters, CKD273, a urinary proteomics-based classifier and its peptides were quantified. The baseline values were analyzed with regard to the clinical parameters and the occurrence of death or renal death during follow-up (3.6 years as the main outcome measurements. None of the patients with CKD2730.55. Unsupervised clustering analysis of the CKD273 peptides separated the patients into two main groups differing in CKD associated parameters. Among the 273 biomarkers, peptides derived from serum proteins were relatively increased in patients with lower glomerular filtration rate, while collagen-derived peptides were relatively decreased (p<0.05; Spearman. CKD273 was different in the groups with different renal function (p<0.003. The CKD273 classifier separated CKD patients according to their renal function and informed on the likelihood of experiencing adverse outcome. Recently defined in a large population, CKD273 is the first proteomic-based classifier successfully tested for prognosis of CKD progression in an independent cohort.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

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Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

2016-06-01

Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

Principles of proteome allocation are revealed using proteomic data and genome-scale models

DEFF Research Database (Denmark)

Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.

2016-01-01

to metabolism and fitness. Using proteomics data, we formulated allocation constraints for key proteome sectors in the ME model. The resulting calibrated model effectively computed the "generalist" (wild-type) E. coli proteome and phenotype across diverse growth environments. Across 15 growth conditions......Integrating omics data to refine or make context-specific models is an active field of constraint-based modeling. Proteomics now cover over 95% of the Escherichia coli proteome by mass. Genome-scale models of Metabolism and macromolecular Expression (ME) compute proteome allocation linked...... of these sectors for the general stress response sigma factor sigma(S). Finally, the sector constraints represent a general formalism for integrating omics data from any experimental condition into constraint-based ME models. The constraints can be fine-grained (individual proteins) or coarse-grained (functionally...

Highly efficient human serum filtration with water-soluble nanoporous nanoparticles

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Antonella Pujia

2010-11-01

Full Text Available Antonella Pujia1, Francesco De Angelis1,2, Domenica Scumaci3, Marco Gaspari3, Carlo Liberale1,2, Patrizio Candeloro1, Giovanni Cuda3, Enzo Di Fabrizio1,21BIONEM Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ, Italy; 2IIT, Italian Institute of Technology, Genova, Italy; 3Proteomics and Mass Spectrometry Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ, ItalyBackground: Human serum has the potential to become the most informative source of novel biomarkers, but its study is very difficult due to the incredible complexity of its molecular composition. We describe a novel tool based on biodegradable nanoporous nanoparticles (NPNPs that allows the harvesting of low-molecular-weight fractions of crude human serum or other biofluids. NPNPs with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon. When incubated with a solution, the NPNPs harvest only the molecules small enough to be absorbed into the nanopores. Then they can be recovered by centrifugation and dissolved in water, making the harvested molecules available for further analyses.Results: Fluorescence microscopy, gel electrophoresis, and mass spectrometry were used to show the enrichment of low-molecular-weight fraction of serum under physiological conditions, with a cut-off of 13 kDa and an enrichment factor >50.Conclusion: From these findings, we conclude that ability to tune pore size, combined with the availability of hundreds of biomolecule cross-linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery.Keywords: nanoporous silicon, nanoparticle, biomarker discovery, human serum proteomics, harvesting

Hormesis of specific IgG antibody to rabies virus in serum of mice irradiated with low dose γ-rays

International Nuclear Information System (INIS)

Liu Qingjie; Chen Deqing

1998-01-01

Objective: To explore the effect of low dose ionizing radiation on specific antibody in mouse serum. Methods: Kunming strain male mice, weighing 18-22 g, aged 6-8 weeks, were immunized intraperitoneally with rabies vaccine after exposure to cobalt-60 γ-rays. The specific IgG antibody against rabies virus in mouse serum was measured. Results: (1) The serum levels of specific IgG in mice irradiated with 5-30 cGy γ-rays were significantly elevated in comparison with those in control mice (P<0.01), the optimum stimulating dose being 10 cGy. (2) Exposure to 10 cGy caused significant enhancement and earlier emergence of the peak level of specific IgG in serum. (3) The hormesis of specific IgG to rabies virus induced by 10 cGy γ-rays could last one week at least. Conclusion: Low dose ionizing radiation can enhance the level of specific antibody in mouse serum, and this effect can last for one week at least

PeptideManager: A Peptide Selection Tool for Targeted Proteomic Studies Involving Mixed Samples from Different Species

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Kevin eDemeure

2014-09-01

Full Text Available The search for clinically useful protein biomarkers using advanced mass spectrometry approaches represents a major focus in cancer research. However, the direct analysis of human samples may be challenging due to limited availability, the absence of appropriate control samples, or the large background variability observed in patient material. As an alternative approach, human tumors orthotopically implanted into a different species (xenografts are clinically relevant models that have proven their utility in pre-clinical research. Patient derived xenografts for glioblastoma have been extensively characterized in our laboratory and have been shown to retain the characteristics of the parental tumor at the phenotypic and genetic level. Such models were also found to adequately mimic the behavior and treatment response of human tumors. The reproducibility of such xenograft models, the possibility to identify their host background and perform tumor-host interaction studies, are major advantages over the direct analysis of human samples.At the proteome level, the analysis of xenograft samples is challenged by the presence of proteins from two different species which, depending on tumor size, type or location, often appear at variable ratios. Any proteomics approach aimed at quantifying proteins within such samples must consider the identification of species specific peptides in order to avoid biases introduced by the host proteome. Here, we present an in-house methodology and tool developed to select peptides used as surrogates for protein candidates from a defined proteome (e.g., human in a host proteome background (e.g., mouse, rat suited for a mass spectrometry analysis. The tools presented here are applicable to any species specific proteome, provided a protein database is available. By linking the information from both proteomes, PeptideManager significantly facilitates and expedites the selection of peptides used as surrogates to analyze

In silico proteome analysis to facilitate proteomics experiments using mass spectrometry

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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.

The HUPO proteomics standards initiative--overcoming the fragmentation of proteomics data.

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Hermjakob, Henning

2006-09-01

Proteomics is a key field of modern biomolecular research, with many small and large scale efforts producing a wealth of proteomics data. However, the vast majority of this data is never exploited to its full potential. Even in publicly funded projects, often the raw data generated in a specific context is analysed, conclusions are drawn and published, but little attention is paid to systematic documentation, archiving, and public access to the data supporting the scientific results. It is often difficult to validate the results stated in a particular publication, and even simple global questions like "In which cellular contexts has my protein of interest been observed?" can currently not be answered with realistic effort, due to a lack of standardised reporting and collection of proteomics data. The Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organisation (HUPO), defines community standards for data representation in proteomics to facilitate systematic data capture, comparison, exchange and verification. In this article we provide an overview of PSI organisational structure, activities, and current results, as well as ways to get involved in the broad-based, open PSI process.

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

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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.

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

Science.gov (United States)

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

Technological advances for deciphering the complexity of psychiatric disorders: merging proteomics with cell biology.

Science.gov (United States)

Wesseling, Hendrik; Guest, Paul C; Lago, Santiago G; Bahn, Sabine

2014-08-01

Proteomic studies have increased our understanding of the molecular pathways affected in psychiatric disorders. Mass spectrometry and two-dimensional gel electrophoresis analyses of post-mortem brain samples from psychiatric patients have revealed effects on synaptic, cytoskeletal, antioxidant and mitochondrial protein networks. Multiplex immunoassay profiling studies have found alterations in hormones, growth factors, transport and inflammation-related proteins in serum and plasma from living first-onset patients. Despite these advances, there are still difficulties in translating these findings into platforms for improved treatment of patients and for discovery of new drugs with better efficacy and side effect profiles. This review describes how the next phase of proteomic investigations in psychiatry should include stringent replication studies for validation of biomarker candidates and functional follow-up studies which can be used to test the impact on physiological function. All biomarker candidates should now be tested in series with traditional and emerging cell biological approaches. This should include investigations of the effects of post-translational modifications, protein dynamics and network analyses using targeted proteomic approaches. Most importantly, there is still an urgent need for development of disease-relevant cellular models for improved translation of proteomic findings into a means of developing novel drug treatments for patients with these life-altering disorders.

Making proteomics data accessible and reusable: current state of proteomics databases and repositories.

Science.gov (United States)

Perez-Riverol, Yasset; Alpi, Emanuele; Wang, Rui; Hermjakob, Henning; Vizcaíno, Juan Antonio

2015-03-01

Compared to other data-intensive disciplines such as genomics, public deposition and storage of MS-based proteomics, data are still less developed due to, among other reasons, the inherent complexity of the data and the variety of data types and experimental workflows. In order to address this need, several public repositories for MS proteomics experiments have been developed, each with different purposes in mind. The most established resources are the Global Proteome Machine Database (GPMDB), PeptideAtlas, and the PRIDE database. Additionally, there are other useful (in many cases recently developed) resources such as ProteomicsDB, Mass Spectrometry Interactive Virtual Environment (MassIVE), Chorus, MaxQB, PeptideAtlas SRM Experiment Library (PASSEL), Model Organism Protein Expression Database (MOPED), and the Human Proteinpedia. In addition, the ProteomeXchange consortium has been recently developed to enable better integration of public repositories and the coordinated sharing of proteomics information, maximizing its benefit to the scientific community. Here, we will review each of the major proteomics resources independently and some tools that enable the integration, mining and reuse of the data. We will also discuss some of the major challenges and current pitfalls in the integration and sharing of the data. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product.

Science.gov (United States)

Matsumoto, Takayuki; Hess, Sonja; Kajiyama, Hiroshi; Sakairi, Toru; Saleem, Moin A; Mathieson, Peter W; Nojima, Yoshihisa; Kopp, Jeffrey B

2010-10-01

The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of factor-binding protein-related protein-1 (IGFBP-rP1), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-β1 stimulation increased IGFBP-rP1 in conditioned medium. We analyzed IGFBP-rP1 glomerular expression in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteome may identify biomarkers of podocyte injury.

Personalized medicine beyond genomics: alternative futures in big data-proteomics, environtome and the social proteome.

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Özdemir, Vural; Dove, Edward S; Gürsoy, Ulvi K; Şardaş, Semra; Yıldırım, Arif; Yılmaz, Şenay Görücü; Ömer Barlas, I; Güngör, Kıvanç; Mete, Alper; Srivastava, Sanjeeva

2017-01-01

No field in science and medicine today remains untouched by Big Data, and psychiatry is no exception. Proteomics is a Big Data technology and a next generation biomarker, supporting novel system diagnostics and therapeutics in psychiatry. Proteomics technology is, in fact, much older than genomics and dates to the 1970s, well before the launch of the international Human Genome Project. While the genome has long been framed as the master or "elite" executive molecule in cell biology, the proteome by contrast is humble. Yet the proteome is critical for life-it ensures the daily functioning of cells and whole organisms. In short, proteins are the blue-collar workers of biology, the down-to-earth molecules that we cannot live without. Since 2010, proteomics has found renewed meaning and international attention with the launch of the Human Proteome Project and the growing interest in Big Data technologies such as proteomics. This article presents an interdisciplinary technology foresight analysis and conceptualizes the terms "environtome" and "social proteome". We define "environtome" as the entire complement of elements external to the human host, from microbiome, ambient temperature and weather conditions to government innovation policies, stock market dynamics, human values, political power and social norms that collectively shape the human host spatially and temporally. The "social proteome" is the subset of the environtome that influences the transition of proteomics technology to innovative applications in society. The social proteome encompasses, for example, new reimbursement schemes and business innovation models for proteomics diagnostics that depart from the "once-a-life-time" genotypic tests and the anticipated hype attendant to context and time sensitive proteomics tests. Building on the "nesting principle" for governance of complex systems as discussed by Elinor Ostrom, we propose here a 3-tiered organizational architecture for Big Data science such as

Unraveling the Molecular Mechanism(s) Underlying Er+/PR-Breast Tumorigenesis Using a Novel Genetically Engineered Mouse Model

Science.gov (United States)

2011-11-01

and subjected to silver stain, immunoblot, or LC-MS/MS spec- tral analyses (The MSU Proteomics Facility, Michigan State University). Mouse Hepatocyte... flotation gradient essentially as described [29]. Briefly, cells were incubated with 10 ng/ml EGF for 10 min at 37uC. After several washings, cells were

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

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Mora-Cartín, Ricardo; Chacón-Díaz, Carlos; Gutiérrez-Jiménez, Cristina; Gurdián-Murillo, Stephany; Lomonte, Bruno; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; Moreno, Edgardo

2016-06-01

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Proteomics in medical microbiology.

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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.

NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

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Saeko Yanaka

2017-09-01

Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers.

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Thelin, Eric Peter; Just, David; Frostell, Arvid; Häggmark-Månberg, Anna; Risling, Mårten; Svensson, Mikael; Nilsson, Peter; Bellander, Bo-Michael

2018-03-15

The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Sprague Dawley rats (n=73) received a 3mm deep controlled cortical impact ("severe injury"). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Identifying Predictors of Taxane-Induced Peripheral Neuropathy Using Mass Spectrometry-Based Proteomics Technology.

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Emily I Chen

Full Text Available Major advances in early detection and therapy have significantly increased the survival of breast cancer patients. Unfortunately, most cancer therapies are known to carry a substantial risk of adverse long-term treatment-related effects. Little is known about patient susceptibility to severe side effects after chemotherapy. Chemotherapy-induced peripheral neuropathy (CIPN is a common side effect of taxanes. Recent advances in genome-wide genotyping and sequencing technologies have supported the discoveries of a number of pharmacogenetic markers that predict response to chemotherapy. However, effectively implementing these pharmacogenetic markers in the clinic remains a major challenge. On the other hand, recent advances in proteomic technologies incorporating mass spectrometry (MS for biomarker discovery show great promise to provide clinically relevant protein biomarkers. In this study, we evaluated the association between protein content in serum exosomes and severity of CIPN. Women with early stage breast cancer receiving adjuvant taxane chemotherapy were assessed with the FACT-Ntx score and serum was collected before and after the taxane treatment. Based on the change in FACT-Ntx score from baseline to 12 month follow-up, we separated patients into two groups: those who had no change (Group 1, N = 9 and those who had a ≥20% worsening (Group 1, N = 8. MS-based proteomics technology was used to identify proteins present in serum exosomes to determine potential biomarkers. Mann-Whitney-Wilcoxon analysis was applied and maximum FDR was controlled at 20%. From the serum exosomes derived from this cohort, we identified over 700 proteins known to be in different subcellular locations and have different functions. Statistical analysis revealed a 12-protein signature that resulted in a distinct separation between baseline serum samples of both groups (q<0.2 suggesting that the baseline samples can predict subsequent neurotoxicity. These toxicity

Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer

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Abedini F

2012-07-01

Full Text Available Fatemeh Abedini,1 Hossein Hosseinkhani,2 Maznah Ismail,1,3 Abraham J Domb,4 Abdul Rahman Omar,1,5 Pei Pei Chong,1,2 Po-Da Hong,3 Dah-Shyong Yu,6 Ira-Yudovin Farber41Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor, 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, 3Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 5Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia, 6Nanomedicine Research Center, National Defense Medical Center, Taipei, TaiwanPurpose: The failure of colorectal cancer treatments is partly due to overexpression of CXCR4 by tumor cells, which plays a critical role in cell metastasis. Moreover, serum alkaline phosphatase (ALP levels are frequently elevated in patients with metastatic colorectal cancer. A polysaccharide, dextran, was chosen as the vector of siRNA. Spermine was conjugated to oxidized dextran by reductive amination process to obtain cationized dextran, so-called dextran-spermine, in order to prepare CXCR4-siRNAs/dextran-spermine nanoparticles. The fabricated nanoparticles were used in order to investigate whether downregulation of CXCR4 expression could affect serum ALP in mouse models of colorectal cancer.Methods: Colorectal cancer was established in BALB/C mice following injection of mouse colon carcinoma cells CT.26WT through the tail vein. CXCR4 siRNA for two sites of the target gene was administered following injection of naked siRNA or siRNA encapsulated into nanoparticles.Results: In vivo animal data revealed that CXCR4 silencing by dextran-spermine nanoparticles significantly downregulated CXCR4 expression compared with naked CXCR4 siRNA. Furthermore, there was

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

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Nie, Song [Biological Sciences Division; Shi, Tujin [Biological Sciences Division; Fillmore, Thomas L. [Biological Sciences Division; Schepmoes, Athena A. [Biological Sciences Division; Brewer, Heather [Biological Sciences Division; Gao, Yuqian [Biological Sciences Division; Song, Ehwang [Biological Sciences Division; Wang, Hui [Biological Sciences Division; Rodland, Karin D. [Biological Sciences Division; Qian, Wei-Jun [Biological Sciences Division; Smith, Richard D. [Biological Sciences Division; Liu, Tao [Biological Sciences Division

2017-08-11

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

DEFF Research Database (Denmark)

Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna

2015-01-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass...

Proteomic analysis of liver in rats chronically exposed to fluoride.

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Heloísa Aparecida Barbosa da Silva Pereira

Full Text Available Fluoride (F is a potent anti-cariogenic element, but when ingestion is excessive, systemic toxicity may be observed. This can occur as acute or chronic responses, depending on both the amount of F and the time of exposure. The present study identified the profile of protein expression possibly associated with F-induced chronic hepatotoxicity. Weanling male Wistar rats (three-weeks old were divided into three groups and treated with drinking water containing 0, 5 or 50 mg/L F for 60 days (n=6/group. At this time point, serum and livers were collected for F analysis, which was done using the ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion. Livers were also submitted to histological and proteomic analyses (2D-PAGE followed by LC-MS/MS. Western blotting was done for confirmation of the proteomic data A dose-response was observed in serum F levels. In the livers, F levels were significantly increased in the 50 mg/L F group compared to groups treated with 0 and 5 mg/L F. Liver morphometric analysis did not reveal alterations in the cellular structures and lipid droplets were present in all groups. Proteomic quantitative intensity analysis detected 33, 44, and 29 spots differentially expressed in the comparisons between control vs. 5 mg/L F, control vs. 50 mg/L F, and 5 mg/L vs. 50 mg/L F, respectively. From these, 92 proteins were successfully identified. In addition, 18, 1, and 5 protein spots were shown to be exclusive in control, 5, and 50 mg/L F, respectively. Most of proteins were related to metabolic process and pronounced alterations were seen for the high-F level group. In F-treated rats, changes in the apolipoprotein E (ApoE and GRP-78 expression may account for the F-induced toxicity in the liver. This can contribute to understanding the molecular mechanisms underlying hepatoxicity induced by F, by indicating key-proteins that should be better addressed in future studies.

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

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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].

Serum proteomic analysis reveals potential serum biomarkers for occupational medicamentosa-like dermatitis caused by trichloroethylene.

Science.gov (United States)

Huang, Peiwu; Ren, Xiaohu; Huang, Zhijun; Yang, Xifei; Hong, Wenxu; Zhang, Yanfang; Zhang, Hang; Liu, Wei; Huang, Haiyan; Huang, Xinfeng; Wu, Desheng; Yang, Linqing; Tang, Haiyan; Zhou, Li; Li, Xuan; Liu, Jianjun

2014-08-17

Trichloroethylene (TCE) is an industrial solvent with widespread occupational exposure and also a major environmental contaminant. Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become one major hazard in China. In this study, sera from 3 healthy controls and 3 OMLDT patients at different disease stages were used for a screening study by 2D-DIGE and MALDI-TOF-MS/MS. Eight proteins including transthyretin (TTR), retinol binding protein 4 (RBP4), haptoglobin, clusterin, serum amyloid A protein (SAA), apolipoprotein A-I, apolipoprotein C-III and apolipoprotein C-II were found to be significantly altered among the healthy, acute-stage, healing-stage and healed-stage groups. Specifically, the altered expression of TTR, RBP4 and haptoglobin were further validated by Western blot analysis and ELISA. Our data not only suggested that TTR, RBP4 and haptoglobin could serve as potential serum biomarkers of OMLDT, but also indicated that measurement of TTR, RBP4 and haptoglobin or their combination could help aid in the diagnosis, monitoring the progression and therapy of the disease. Copyright © 2014. Published by Elsevier Ireland Ltd.

Bacterial membrane proteomics.

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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.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA

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Malathi Narayan

2016-06-01

Full Text Available Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA on a mouse microglial (N9 cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003032.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome

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Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

2016-01-01

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed...... by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly...

Translational plant proteomics: a perspective.

Science.gov (United States)

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.

Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins.

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Brick, Tabea; Ege, Markus; Boeren, Sjef; Böck, Andreas; von Mutius, Erika; Vervoort, Jacques; Hettinga, Kasper

2017-08-31

Consumption of raw cow's milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general is missing. The aim of this study was to compare the native milk serum proteome between raw cow's milk and various industrially applied processing methods, i.e., homogenization, fat separation, pasteurization, ultra-heat treatment (UHT), treatment for extended shelf-life (ESL), and conventional boiling. Each processing method was applied to the same three pools of raw milk. Levels of detectable proteins were quantified by liquid chromatography/tandem mass spectrometry following filter aided sample preparation. In total, 364 milk serum proteins were identified. The 140 proteins detectable in 66% of all samples were entered in a hierarchical cluster analysis. The resulting proteomics pattern separated mainly as high (boiling, UHT, ESL) versus no/low heat treatment (raw, skimmed, pasteurized). Comparing these two groups revealed 23 individual proteins significantly reduced by heating, e.g., lactoferrin (log2-fold change = -0.37, p = 0.004), lactoperoxidase (log2-fold change = -0.33, p = 0.001), and lactadherin (log2-fold change = -0.22, p = 0.020). The abundance of these heat sensitive proteins found in higher quantity in native cow's milk compared to heat treated milk, renders them potential candidates for protection from asthma, allergies, and respiratory infections.

Proteomics in Argentina - limitations and future perspectives: A special emphasis on meat proteomics.

Science.gov (United States)

Fadda, Silvina; Almeida, André M

2015-11-01

Argentina is one of the most relevant countries in Latin America, playing a major role in regional economics, culture and science. Over the last 80 years, Argentinean history has been characterized by several upward and downward phases that had major consequences on the development of science in the country and most recently on proteomics. In this article, we characterize the evolution of Proteomics sciences in Argentina over the last decade and a half. We describe the proteomics publication output of the country in the framework of the regional and international contexts, demonstrating that Argentina is solidly anchored in a regional context, showing results similar to other emergent and Latin American countries, albeit still far from the European, American or Australian realities. We also provide a case-study on the importance of Proteomics to a specific sector in the area of food science: the use of bacteria of technological interest, highlighting major achievements obtained by Argentinean proteomics scientists. Finally, we provide a general picture of the endeavors being undertaken by Argentinean Proteomics scientists and their international collaborators to promote the Proteomics-based research with the new generation of scientists and PhD students in both Argentina and other countries in the Southern cone. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteome Characteristics of Non-Alcoholic Steatohepatitis Liver Tissue and Associated Hepatocellular Carcinomas

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Anna Kakehashi

2017-02-01

Full Text Available To uncover mechanisms of nonalcoholic steatohepatitis (NASH associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs and HCCs of HCV+ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV+ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor β, SMAD family member 3, β-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1, and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

NARCIS (Netherlands)

Zanivan, S.; Meves, A.; Behrendt, K.; Schoof, E.M.; Neilson, L.J.; Cox, J.; Tang, H.R.; Kalna, G.; Ree, J.H. van; Deursen, J.M.A. van; Trempus, C.S.; Machesky, L.M.; Linding, R.; Wickstrom, S.A.; Fassler, R.; Mann, M.

2013-01-01

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic

Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification.

Science.gov (United States)

van Rooden, Eva J; Florea, Bogdan I; Deng, Hui; Baggelaar, Marc P; van Esbroeck, Annelot C M; Zhou, Juan; Overkleeft, Herman S; van der Stelt, Mario

2018-04-01

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

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

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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.

Serum IGF-1 affects skeletal acquisition in a temporal and compartment-specific manner.

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Hayden-William Courtland

2011-03-01

Full Text Available Insulin-like growth factor-1 (IGF-1 plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD. Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID, in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice prior to adulthood (4 weeks decreased trabecular bone architecture and significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th by 16 weeks (adulthood. Likewise, depletion of serum IGF-1 in iLID males at 8 weeks of age, resulted in significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th by 32 weeks (late adulthood, but had no effect on trabecular bone architecture. In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks resulted in enhancement of trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase, depletion of IGF-1 after peak bone acquisition (16 weeks is compartment-specific and does not have a detrimental effect on cortical bone mass in the older adult mouse.

Unlocking biomarker discovery: large scale application of aptamer proteomic technology for early detection of lung cancer.

Directory of Open Access Journals (Sweden)

Rachel M Ostroff

Full Text Available BACKGROUND: Lung cancer is the leading cause of cancer deaths worldwide. New diagnostics are needed to detect early stage lung cancer because it may be cured with surgery. However, most cases are diagnosed too late for curative surgery. Here we present a comprehensive clinical biomarker study of lung cancer and the first large-scale clinical application of a new aptamer-based proteomic technology to discover blood protein biomarkers in disease. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a multi-center case-control study in archived serum samples from 1,326 subjects from four independent studies of non-small cell lung cancer (NSCLC in long-term tobacco-exposed populations. Sera were collected and processed under uniform protocols. Case sera were collected from 291 patients within 8 weeks of the first biopsy-proven lung cancer and prior to tumor removal by surgery. Control sera were collected from 1,035 asymptomatic study participants with ≥ 10 pack-years of cigarette smoking. We measured 813 proteins in each sample with a new aptamer-based proteomic technology, identified 44 candidate biomarkers, and developed a 12-protein panel (cadherin-1, CD30 ligand, endostatin, HSP90α, LRIG3, MIP-4, pleiotrophin, PRKCI, RGM-C, SCF-sR, sL-selectin, and YES that discriminates NSCLC from controls with 91% sensitivity and 84% specificity in cross-validated training and 89% sensitivity and 83% specificity in a separate verification set, with similar performance for early and late stage NSCLC. CONCLUSIONS/SIGNIFICANCE: This study is a significant advance in clinical proteomics in an area of high unmet clinical need. Our analysis exceeds the breadth and dynamic range of proteome interrogated of previously published clinical studies of broad serum proteome profiling platforms including mass spectrometry, antibody arrays, and autoantibody arrays. The sensitivity and specificity of our 12-biomarker panel improves upon published protein and gene expression panels

[Methods of quantitative proteomics].

Science.gov (United States)

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.

Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

Science.gov (United States)

Kim, Thomas D.; Craig, Paul A.

2010-01-01

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

Translational plant proteomics: A perspective

NARCIS (Netherlands)

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

Evolution of Clinical Proteomics and its Role in Medicine | Office of Cancer Clinical Proteomics Research

Science.gov (United States)

NCI's Office of Cancer Clinical Proteomics Research authored a review of the current state of clinical proteomics in the peer-reviewed Journal of Proteome Research. The review highlights outcomes from the CPTC program and also provides a thorough overview of the different technologies that have pushed the field forward. Additionally, the review provides a vision for moving the field forward through linking advances in genomic and proteomic analysis to develop new, molecularly targeted interventions.

Efficacy of Enrofloxacin in a Mouse Model of Sepsis

OpenAIRE

Slate, Andrea R; Bandyopadhyay, Sheila; Francis, Kevin P; Papich, Mark G; Karolewski, Brian; Hod, Eldad A; Prestia, Kevin A

2014-01-01

We examined the efficacy of enrofloxacin administered by 2 different routes in a mouse model of sepsis. Male CD1 mice were infected with a bioluminescent strain of enteropathogenic Escherichia coli and treated with enrofloxacin either by injection or in drinking water. Peak serum levels were evaluated by using HPLC. Mice were monitored for signs of clinical disease, and infections were monitored by using bioluminescence imaging. Serum levels of enrofloxacin and the active metabolite ciproflox...

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

Science.gov (United States)

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.

Analysis of human serum by liquid chromatography-mass spectrometry: improved sample preparation and data analysis.

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Govorukhina, N I; Reijmers, T H; Nyangoma, S O; van der Zee, A G J; Jansen, R C; Bischoff, R

2006-07-07

Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC-MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC-MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC-MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC-MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C18 column and reversed-phase LC-MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: <0.1% RSD; peak area: <30% RSD). Application of an LC-MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart

Proteomics of Maize Root Development.

Science.gov (United States)

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.

Proteomics of Maize Root Development

Directory of Open Access Journals (Sweden)

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.

Proteomic approach for identifying gonad differential proteins in the oyster (Crassostrea angulata) following food-chain contamination with HgCl2.

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Zhang, Qing-Hong; Huang, Lin; Zhang, Yong; Ke, Cai-Huan; Huang, He-Qing

2013-12-06

Hg discharged into the environmental waters can generally be bioaccumulated, transformed and transmited by living organisms, thus resulting in the formation of Hg-toxicity food chains. The pathway and toxicology of food chain contaminated with environmental Hg are rarely revealed by proteomics. Here, we showed that differential proteomics had the potential to understand reproduction toxicity mechanism in marine molluscs through the Hg-contaminated food chain. Hg bioaccumulation was found in every link of the HgCl2-Chlorella vulgaris-oyster-mice food chain. Morphological observations identified the lesions in both the oyster gonad and the mice ovary. Differential proteomics was used to study the mechanisms of Hg toxicity in the oyster gonad and to find some biomarkers of Hg contamination in food chain. Using 2-DE and MALDI-TOF/TOF MS, we identified 13 differential protein spots, of which six were up-regulated, six were down-regulated, while one was undecided. A portion of these differential proteins was further confirmed using real-time PCR and western blotting methods. Their major functions involved binding, protein translocation, catalysis, regulation of energy metabolism, reproductive functioning and structural molecular activity. Among these proteins, 14-3-3 protein, GTP binding protein, arginine kinase (AK) and 71kDa heat shock connate protein (HSCP 71) are considered to be suitable biomarkers of environmental Hg contamination. Furthermore, we established the gene correspondence, responding to Hg reproductive toxicity, between mouse and oyster, and then used real-time PCR to analyze mRNA differential expression of the corresponding genes in mice. The results indicated that the mechanism of Hg reproductive toxicity in mouse was similar to that in oyster. We suggest that the proteomics would be further developed in application research of food safety including toxicological mechanism. It is well known that mercury (Hg) is one of the best toxic metal elements in

Two different mechanisms of immune-complex trapping in the mouse spleen during immune responses

NARCIS (Netherlands)

Yoshida, K.; van den Berg, T. K.; Dijkstra, C. D.

1993-01-01

The capacity of immune-complex (IC) trapping was examined using purified horse radish peroxidase (HRP)-anti-HRP (PAP) on frozen sections of mouse spleen in vitro. We investigated the trapping mechanisms by applying the IC with or without fresh mouse serum added on the spleen sections of naive as

Integrating cell biology and proteomic approaches in plants.

Science.gov (United States)

Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

2017-10-03

Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of

Genomes to Proteomes

Energy Technology Data Exchange (ETDEWEB)

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

Comparative proteome analysis of three mouse lung adenocarcinoma CMT cell lines with different metastatic potential by two-dimensional gel electrophoresis and mass spectrometry.

Science.gov (United States)

Zhang, Kelan; Wrzesinski, Krzysztof; Stephen, J Fey; Larsen, Peter Mose; Zhang, Xumin; Roepstorff, Peter

2008-12-01

Metastasis is a lethal attribute of a cancer and presents a continuing therapeutic challenge. Metastasis is a highly complex process and more knowledge about the mechanisms behind metastasis is highly desirable. Isogenic CMT cell lines were selected from a spontaneous mouse lung adenocarcinoma and characterized in vivo to have different metastatic potential. In this study, the comprehensive protein expression profiles of three of these CMT cell lines at passage 5, 15 and 35 were analyzed by 2-DE separation followed by MS identification. As a result, 82 and 40 unique proteins were found to be significantly up- or down-regulated between cell lines with different metastatic potential at passages 5 and 15, respectively. These proteins were identified by MS and most of them have previously been reported to be related to cancer development and/or metastasis. Bioinformatics analysis indicated that several of the proteins were involved in proteasome, cell-cycle and cell-communication pathways. Among them, some keratins, 14-3-3 proteins and 26S proteasome proteins were identified and their aberrant expression may be directly or indirectly involved in cancer development and metastasis. In conclusion, our comprehensive 2-DE-based proteomics studies revealed some candidate proteins, protein families and signaling pathways, which might be important in cancer development and metastasis.

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

DEFF Research Database (Denmark)

Young, Jette Feveile; Larsen, Lotte Bach; Malmendal, Anders

2010-01-01

Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat......-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating...... the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. METHODS: Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension...

Proteomic explorations of autism spectrum disorder.

Science.gov (United States)

Szoko, Nicholas; McShane, Adam J; Natowicz, Marvin R

2017-09-01

Proteomics, the large-scale study of protein expression in cells and tissues, is a powerful tool to study the biology of clinical conditions and has provided significant insights in many experimental systems. Herein, we review the basics of proteomic methodology and discuss challenges in using proteomic approaches to study autism. Unlike other experimental approaches, such as genomic approaches, there have been few large-scale studies of proteins in tissues from persons with autism. Most of the proteomic studies on autism used blood or other peripheral tissues; few studies used brain tissue. Some studies found dysregulation of aspects of the immune system or of aspects of lipid metabolism, but no consistent findings were noted. Based on the challenges in using proteomics to study autism, we discuss considerations for future studies. Apart from the complex technical considerations implicit in any proteomic analysis, key nontechnical matters include attention to subject and specimen inclusion/exclusion criteria, having adequate sample size to ensure appropriate powering of the study, attention to the state of specimens prior to proteomic analysis, and the use of a replicate set of specimens, when possible. We conclude by discussing some potentially productive uses of proteomics, potentially coupled with other approaches, for future autism research including: (1) proteomic analysis of banked human brain specimens; (2) proteomic analysis of tissues from animal models of autism; and (3) proteomic analysis of induced pluripotent stem cells that are differentiated into various types of brain cells and neural organoids. Autism Res 2017, 10: 1460-1469. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Proteomics dataset

DEFF Research Database (Denmark)

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....

A Hybrid Feature Subset Selection Algorithm for Analysis of High Correlation Proteomic Data

Science.gov (United States)

Kordy, Hussain Montazery; Baygi, Mohammad Hossein Miran; Moradi, Mohammad Hassan

2012-01-01

Pathological changes within an organ can be reflected as proteomic patterns in biological fluids such as plasma, serum, and urine. The surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been used to generate proteomic profiles from biological fluids. Mass spectrometry yields redundant noisy data that the most data points are irrelevant features for differentiating between cancer and normal cases. In this paper, we have proposed a hybrid feature subset selection algorithm based on maximum-discrimination and minimum-correlation coupled with peak scoring criteria. Our algorithm has been applied to two independent SELDI-TOF MS datasets of ovarian cancer obtained from the NCI-FDA clinical proteomics databank. The proposed algorithm has used to extract a set of proteins as potential biomarkers in each dataset. We applied the linear discriminate analysis to identify the important biomarkers. The selected biomarkers have been able to successfully diagnose the ovarian cancer patients from the noncancer control group with an accuracy of 100%, a sensitivity of 100%, and a specificity of 100% in the two datasets. The hybrid algorithm has the advantage that increases reproducibility of selected biomarkers and able to find a small set of proteins with high discrimination power. PMID:23717808

Streptococcus iniae SF1: complete genome sequence, proteomic profile, and immunoprotective antigens.

Directory of Open Access Journals (Sweden)

Bao-cun Zhang

Full Text Available Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS, 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control.

Streptococcus iniae SF1: Complete Genome Sequence, Proteomic Profile, and Immunoprotective Antigens

Science.gov (United States)

Zhang, Bao-cun; Zhang, Jian; Sun, Li

2014-01-01

Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS), 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control. PMID:24621602

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant

Directory of Open Access Journals (Sweden)

Lina Zhang

2016-06-01

Full Text Available Here we provide data from shot-gun proteomics, using filtered-aided sample preparation (FASP, dimethyl labeling and LC–MS/MS, to quantify the changes in the repertoire of human milk proteins over lactation. Milk serum proteins were analyzed at week 1, 2, 3 4, 8, 16, and 24 in milk from four individual mothers. A total of 247 proteins were identified, of which 200 proteins were quantified. The data supplied in this article supports the accompanying publication (Zhang et al., 2006 [1]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2016 [2] via the PRIDE partner repository with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003465.

DNA topoisomerase II enzyme activity appears in mouse sperm ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... 4 mg/ml bovine serum albumin (BSA; Sigma, Chemical Co., U.S.A) and at time of use, it was ..... Differential growth of mouse preimplantation embryos in chemically ... I. In vitro fertilization of eggs by fresh epididymal sperms.

Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

International Nuclear Information System (INIS)

Miura, Taichi; Hirano, Kazumi; Ogura, Chika; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Ando, Ayumi; Kanazawa, Tatsuya; Hamaguchi, Satoshi

2014-01-01

Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H 2 O 2 ), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells. (paper)

A systematic evaluation of normalization methods in quantitative label-free proteomics.

Science.gov (United States)

Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

2018-01-01

To date, mass spectrometry (MS) data remain inherently biased as a result of reasons ranging from sample handling to differences caused by the instrumentation. Normalization is the process that aims to account for the bias and make samples more comparable. The selection of a proper normalization method is a pivotal task for the reliability of the downstream analysis and results. Many normalization methods commonly used in proteomics have been adapted from the DNA microarray techniques. Previous studies comparing normalization methods in proteomics have focused mainly on intragroup variation. In this study, several popular and widely used normalization methods representing different strategies in normalization are evaluated using three spike-in and one experimental mouse label-free proteomic data sets. The normalization methods are evaluated in terms of their ability to reduce variation between technical replicates, their effect on differential expression analysis and their effect on the estimation of logarithmic fold changes. Additionally, we examined whether normalizing the whole data globally or in segments for the differential expression analysis has an effect on the performance of the normalization methods. We found that variance stabilization normalization (Vsn) reduced variation the most between technical replicates in all examined data sets. Vsn also performed consistently well in the differential expression analysis. Linear regression normalization and local regression normalization performed also systematically well. Finally, we discuss the choice of a normalization method and some qualities of a suitable normalization method in the light of the results of our evaluation. © The Author 2016. Published by Oxford University Press.

1001 Proteomes: a functional proteomics portal for the analysis of Arabidopsis thaliana accessions.

Science.gov (United States)

Joshi, Hiren J; Christiansen, Katy M; Fitz, Joffrey; Cao, Jun; Lipzen, Anna; Martin, Joel; Smith-Moritz, A Michelle; Pennacchio, Len A; Schackwitz, Wendy S; Weigel, Detlef; Heazlewood, Joshua L

2012-05-15

The sequencing of over a thousand natural strains of the model plant Arabidopsis thaliana is producing unparalleled information at the genetic level for plant researchers. To enable the rapid exploitation of these data for functional proteomics studies, we have created a resource for the visualization of protein information and proteomic datasets for sequenced natural strains of A. thaliana. The 1001 Proteomes portal can be used to visualize amino acid substitutions or non-synonymous single-nucleotide polymorphisms in individual proteins of A. thaliana based on the reference genome Col-0. We have used the available processed sequence information to analyze the conservation of known residues subject to protein phosphorylation among these natural strains. The substitution of amino acids in A. thaliana natural strains is heavily constrained and is likely a result of the conservation of functional attributes within proteins. At a practical level, we demonstrate that this information can be used to clarify ambiguously defined phosphorylation sites from phosphoproteomic studies. Protein sets of available natural variants are available for download to enable proteomic studies on these accessions. Together this information can be used to uncover the possible roles of specific amino acids in determining the structure and function of proteins in the model plant A. thaliana. An online portal to enable the community to exploit these data can be accessed at http://1001proteomes.masc-proteomics.org/

The differential plasma proteome of obese and overweight individuals undergoing a nutritional weight loss and maintenance intervention

DEFF Research Database (Denmark)

Oller Moreno, Sergio; Cominetti, Ornella; Núñez Galindo, Antonio

2018-01-01

PURPOSE: The nutritional intervention program "DiOGenes" focuses on how obesity can be prevented and treated from a dietary perspective. We generated differential plasma proteome profiles in the DiOGenes cohort to identify proteins associated with weight loss and maintenance and explore their rel......PURPOSE: The nutritional intervention program "DiOGenes" focuses on how obesity can be prevented and treated from a dietary perspective. We generated differential plasma proteome profiles in the DiOGenes cohort to identify proteins associated with weight loss and maintenance and explore...... with largest changes were sex hormone-binding globulin, adiponectin, C-reactive protein, calprotectin, serum amyloid A, and proteoglycan 4 (PRG4), whose association with obesity and weight loss is known. We identified new putative biomarkers for weight loss/maintenance. Correlation between PRG4 and proline......-rich acidic protein 1 (PRAP1) variation and Matsuda insulin sensitivity increment was showed. CONCLUSIONS AND CLINICAL RELEVANCE: MS-based proteomic analysis of a large cohort of non-diabetic overweight and obese individuals concomitantly identified known and novel proteins associated with weight loss...

Xylem sap proteomics.

Science.gov (United States)

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.

Farm animal proteomics - A review

DEFF Research Database (Denmark)

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...

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

Science.gov (United States)

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

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

Science.gov (United States)

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

Proteomic analysis of human tooth pulp: proteomics of human tooth.

Science.gov (United States)

Eckhardt, Adam; Jágr, Michal; Pataridis, Statis; Mikšík, Ivan

2014-12-01

The unique pulp-dentin complex demonstrates strong regenerative potential, which enables it to respond to disease and traumatic injury. Identifying the proteins of the pulp-dentin complex is crucial to understanding the mechanisms of regeneration, tissue calcification, defense processes, and the reparation of dentin by dental pulp. The lack of knowledge of these proteins limits the development of more efficient therapies. The proteomic profile of human tooth pulp was investigated and compared with the proteome of human dentin and blood. The samples of tooth pulp were obtained from 5 sound permanent human third molars of 5 adults (n = 5). The extracted proteins were separated by 2-dimensional gel electrophoresis, analyzed by nano-liquid chromatography tandem mass spectrometry, and identified by correlating mass spectra to the proteomic databases. A total of 342 proteins were identified with high confidence, and 2 proteins were detected for the first time in an actual human sample. The identified tooth pulp proteins have a variety of functions: structural, catalytic, transporter, protease activity, immune response, and many others. In a comparison with dentin and blood plasma, 140 (pulp/dentin) shared proteins were identified, 37 of which were not observed in plasma. It can be suggested that they might participate in the unique pulp-dentin complex. This proteomic investigation of human tooth pulp, together with the previously published study of human dentin, is one of the most comprehensive proteome lists of human teeth to date. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Peptidome profiling of human serum of uveal melanoma patients based on magnetic bead fractionation and mass spectrometry

Directory of Open Access Journals (Sweden)

Xiang-Yu Shi

2017-06-01

Full Text Available AIM: To find new biomarkers for uveal melanoma (UM by analyzing the serum peptidome profile. METHODS: Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometer were used to compile serum peptide profiles. RESULTS: A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity (83.3%, specificity (100% and accuracy rate (93.0% together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen α-chain precursors were identified as potential UM markers. CONCLUSION: We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI-TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.

Mining the granule proteome

DEFF Research Database (Denmark)

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...

Advances of Proteomic Sciences in Dentistry.

Science.gov (United States)

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-05-13

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.

Proteomics - new analytical approaches

International Nuclear Information System (INIS)

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

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko

2017-05-10

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

Science.gov (United States)

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-06-23

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-01-01

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

The proteome of human saliva

Science.gov (United States)

Griffin, Timothy J.

2013-05-01

Human saliva holds tremendous potential for transforming disease and health diagnostics given its richness of molecular information and non-invasive collection. Enumerating its molecular constituents is an important first step towards reaching this potential. Among the molecules in saliva, proteins and peptides arguably have the most value: they can directly indicate biochemical functions linked to a health condition/disease state, and they are attractive targets for biomarker assay development. However, cataloging and defining the human salivary proteome is challenging given the dynamic, chemically heterogeneous and complex nature of the system. In addition, the overall human saliva proteome is composed of several "sub-proteomes" which include: intact full length proteins, proteins carrying post-translational modifications (PTMs), low molecular weight peptides, and the metaproteome, derived from protein products from nonhuman organisms (e.g. microbes) present in the oral cavity. Presented here will be a summary of communal efforts to meet the challenge of characterizing the multifaceted saliva proteome, focusing on the use of mass spectrometry as the proteomic technology of choice. Implications of these efforts to characterize the salivary proteome in the context of disease diagnostics will also be discussed.

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

DEFF Research Database (Denmark)

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...

Inhibiting Effects of Achyranthes Bidentata Polysaccharide and Lycium Barbarum Polysaccharide on Nonenzyme Glycation in D-galactose Induced Mouse Aging Model

Institute of Scientific and Technical Information of China (English)

HONG-BIN DENG; DA-PENG CUI; JIAN-MING JIANG; YAN-CHUN FENG; NIAN-SHENG CAI; DIAN-DONG LI

2003-01-01

To investigate the inhibiting effects and mechanism of achyranthes bidentata polysaccharide (ABP) and lycium barbarum polysaccharide (LBP) on nonenzyme glycation in D-galactose induced mouse aging model. Methods Serum AGE levels were determined by AGE-ELISA, MTT method was used to determine lymphocyte proliferation, IL-2 activity was determined by a bioassay method. Spontaneous motor activity was used to detect mouse's neuromuscular movement, latency of step-through method was used to examine learning and memory abilities of mouse, colormetric assay was used to determine hydroxyproline concentration in mouse skin, pyrogallol autoxidation method was used to determine superoxide dismutase (SOD) activity of erythrocytes. Results Decreased levels of serum AGE, hydroxyproline concentration in mouse skin and spontaneous motor activity in D-galactose mouse aging model were detected after treated with ABP or LBP, while lymphocyte proliferation and IL-2 activity, learning and memory abilities,SOD activity of erythrocytes, were enhanced. Conclusions ABP and LBP could inhibit nonenzyme glycation in D-galactose induced mouse aging model in vivo and ABP has a better inhibiting effect than LBP.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

KAUST Repository

Marondedze, Claudius; Wong, Aloysius Tze; Groen, Arnoud; Serano, Natalia Lorena Gorron; Jankovic, Boris R.; Lilley, Kathryn; Gehring, Christoph A; Thomas, Ludivine

2014-01-01

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

Directory of Open Access Journals (Sweden)

Claudius Marondedze

2014-12-01

Full Text Available The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

KAUST Repository

Marondedze, Claudius

2014-12-31

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Proteomic profiling of antibody-inducing immunogens in tumor tissue identifies PSMA1, LAP3, ANXA3, and maspin as colon cancer markers

Science.gov (United States)

Yang, Qian; Roehrl, Michael H.; Wang, Julia Y.

2018-01-01

We hypothesized that cancer tissue immunogens – antigens capable of inducing specific antibody production in patients – are promising targets for development of precision diagnostics and humoral immunotherapies. We developed an innovative immuno-proteomic strategy and identified new immunogenic markers of colon cancer. Proteins from cancers and matched normal tissues were separated by 2D gel electrophoresis and blotted with serum antibodies from the same patients. Antibody-reactive proteins were sequenced by mass spectrometry and validated by Western blotting and immunohistochemistry. 170 serum antibody-reactive proteins were identified only in cancerous but not matched normal. Among these, proteasome subunit alpha type 1 (PSA1), leucine aminopeptidase 3 (LAP3), annexin A3 (ANXA3), and maspin (serpin B5) were reproducibly found in tissues from three patients. Differential expression patterns were confirmed in samples from eight patients with various stages of colon adenocarcinoma and liver metastases. These tumor-resident proteins and/or their associated serum antibodies may be promising markers for colon cancer screening and early diagnosis. Furthermore, tumor tissue-specific antibodies could potentially be exploited as immunotherapeutic targets against cancer. More generally, proteomic profiling of antibody-inducing cancer-associated immunogens represents a powerful generic method for uncovering the tumor antigen-ome, i.e., the totality of immunogenic tumor-associated proteins. PMID:29423100

Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome.

Directory of Open Access Journals (Sweden)

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.

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

Science.gov (United States)

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.

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

International Nuclear Information System (INIS)

Yu, Qing; Shi, Xudong; Feng, Yu; Kent, K. Craig; Li, Lingjun

2017-01-01

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Energy Technology Data Exchange (ETDEWEB)

Yu, Qing [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Shi, Xudong [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Feng, Yu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Kent, K. Craig [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Li, Lingjun, E-mail: lingjun.li@wisc.edu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Department of Chemistry, University of Wisconsin, Madison, WI 53706 (United States)

2017-05-22

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

Science.gov (United States)

Sinclair, John; Timms, John F

2011-08-01

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography ? Mass Spectrometry

International Nuclear Information System (INIS)

Petyuk, Vladislav A.; Qian, Weijun; Chin, Mark H.; Wang, Haixing H.; Livesay, Eric A.; Monroe, Matthew E.; Adkins, Joshua N.; Jaitly, Navdeep; Anderson, David J.; Camp, David G.; Smith, Desmond J.; Smith, Richard D.

2007-01-01

Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a ''universal'' stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion

Network-based analysis of proteomic profiles

KAUST Repository

Wong, Limsoon

2016-01-26

Mass spectrometry (MS)-based proteomics is a widely used and powerful tool for profiling systems-wide protein expression changes. It can be applied for various purposes, e.g. biomarker discovery in diseases and study of drug responses. Although RNA-based high-throughput methods have been useful in providing glimpses into the underlying molecular processes, the evidences they provide are indirect. Furthermore, RNA and corresponding protein levels have been known to have poor correlation. On the other hand, MS-based proteomics tend to have consistency issues (poor reproducibility and inter-sample agreement) and coverage issues (inability to detect the entire proteome) that need to be urgently addressed. In this talk, I will discuss how these issues can be addressed by proteomic profile analysis techniques that use biological networks (especially protein complexes) as the biological context. In particular, I will describe several techniques that we have been developing for network-based analysis of proteomics profile. And I will present evidence that these techniques are useful in identifying proteomics-profile analysis results that are more consistent, more reproducible, and more biologically coherent, and that these techniques allow expansion of the detected proteome to uncover and/or discover novel proteins.

Arabidopsis peroxisome proteomics

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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.

Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus

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Robert Claude

2011-09-01

Full Text Available Abstract Background Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV, displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231, were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1, as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection. Results Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3, was

Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle

DEFF Research Database (Denmark)

Steinberger, Martin; Föller, Michael; Vogelgesang, Silke

2015-01-01

Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety...... of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber...... size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30–50 %. Muscles from sgk1 -/- mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1...

Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

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Hong-Jhang Chen

Full Text Available Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD, a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877, 1.71% (15/877, and 2.62% (23/877 of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB (Alb, Fga, and Trf, suppressed excitotoxicity (Grm5, Gnai, and Gdi, and enhanced energy metabolism (Bdh, thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3 and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.

Proteomics Insights into Autophagy.

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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.

Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

DEFF Research Database (Denmark)

Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris

2008-01-01

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much...... proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function....

[Progress in stable isotope labeled quantitative proteomics methods].

Science.gov (United States)

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2013-06-01

Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

DEFF Research Database (Denmark)

Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

2012-01-01

Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

A mighty small heart: the cardiac proteome of adult Drosophila melanogaster.

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Anthony Cammarato

2011-04-01

Full Text Available Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca(2+-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25% had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.

PETALS: Proteomic Evaluation and Topological Analysis of a mutated Locus' Signaling

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Patel Vishal

2010-12-01

Full Text Available Abstract Background Colon cancer is driven by mutations in a number of genes, the most notorious of which is Apc. Though much of Apc's signaling has been mechanistically identified over the years, it is not always clear which functions or interactions are operative in a particular tumor. This is confounded by the presence of mutations in a number of other putative cancer driver (CAN genes, which often synergize with mutations in Apc. Computational methods are, thus, required to predict which pathways are likely to be operative when a particular mutation in Apc is observed. Results We developed a pipeline, PETALS, to predict and test likely signaling pathways connecting Apc to other CAN-genes, where the interaction network originating at Apc is defined as a "blossom," with each Apc-CAN-gene subnetwork referred to as a "petal." Known and predicted protein interactions are used to identify an Apc blossom with 24 petals. Then, using a novel measure of bimodality, the coexpression of each petal is evaluated against proteomic (2 D differential In Gel Electrophoresis, 2D-DIGE measurements from the Apc1638N+/-mouse to test the network-based hypotheses. Conclusions The predicted pathways linking Apc and Hapln1 exhibited the highest amount of bimodal coexpression with the proteomic targets, prioritizing the Apc-Hapln1 petal over other CAN-gene pairs and suggesting that this petal may be involved in regulating the observed proteome-level effects. These results not only demonstrate how functional 'omics data can be employed to test in silico predictions of CAN-gene pathways, but also reveal an approach to integrate models of upstream genetic interference with measured, downstream effects.

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

Science.gov (United States)

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

NIH Common Fund - Disruptive Proteomics Technologies - Challenges and Opportunities | Office of Cancer Clinical Proteomics Research

Science.gov (United States)

This Request for Information (RFI) is directed toward determining how best to accelerate research in disruptive proteomics technologies. The Disruptive Proteomics Technologies (DPT) Working Group of the NIH Common Fund wishes to identify gaps and opportunities in current technologies and methodologies related to proteome-wide measurements.  For the purposes of this RFI, “disruptive” is defined as very rapid, very significant gains, similar to the "disruptive" technology development that occurred in DNA sequencing technology.

A decade of proteomics accomplished! Central and Eastern European Proteomic Conference (CEEPC) celebrates its 10th Anniversary in Budapest, Hungary.

Science.gov (United States)

Gadher, Suresh Jivan; Drahos, László; Vékey, Károly; Kovarova, Hana

2017-07-01

The Central and Eastern European Proteomic Conference (CEEPC) proudly celebrated its 10th Anniversary with an exciting scientific program inclusive of proteome, proteomics and systems biology in Budapest, Hungary. Since 2007, CEEPC has represented 'state-of the-art' proteomics in and around Central and Eastern Europe and these series of conferences have become a well-recognized event in the proteomic calendar. Fresher challenges and global healthcare issues such as ageing and chronic diseases are driving clinical and scientific research towards regenerative, reparative and personalized medicine. To this end, proteomics may enable diverse intertwining research fields to reach their end goals. CEEPC will endeavor to facilitate these goals.

Proteome reference map of Drosophila melanogaster head.

Science.gov (United States)

Lee, Tian-Ren; Huang, Shun-Hong; Lee, Chi-Ching; Lee, Hsiao-Yun; Chan, Hsin-Tzu; Lin, Kuo-Sen; Chan, Hong-Lin; Lyu, Ping-Chiang

2012-06-01

Drosophila melanogaster has been used as a genetic model organism to understand the fundamental molecular mechanisms in human biology including memory formation that has been reported involving protein synthesis and/or post-translational modification. In this study, we employed a proteomic platform based on fluorescent 2DE and MALDI-TOF MS to build a standard D. melanogaster head proteome map for proteome-proteome comparison. In order to facilitate the comparison, an interactive database has been constructed for systematically integrating and analyzing the proteomes from different conditions and further implicated to study human diseases related to D. melanogaster model. In summary, the fundamental head proteomic database and bioinformatic analysis will be useful for further elucidating the biological mechanisms such as memory formation and neurodegenerative diseases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.

Science.gov (United States)

Vrana, Julie A; Theis, Jason D; Dasari, Surendra; Mereuta, Oana M; Dispenzieri, Angela; Zeldenrust, Steven R; Gertz, Morie A; Kurtin, Paul J; Grogg, Karen L; Dogan, Ahmet

2014-07-01

Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method for the diagnosis of systemic amyloidosis. Here we describe the development and implementation of a clinical assay using mass spectrometry-based proteomics to type amyloidosis in subcutaneous fat aspirates. First, we validated the assay comparing amyloid-positive (n=43) and -negative (n=26) subcutaneous fat aspirates. The assay classified amyloidosis with 88% sensitivity and 96% specificity. We then implemented the assay as a clinical test, and analyzed 366 amyloid-positive subcutaneous fat aspirates in a 4-year period as part of routine clinical care. The assay had a sensitivity of 90%, and diverse amyloid types, including immunoglobulin light chain (74%), transthyretin (13%), serum amyloid A (%1), gelsolin (1%), and lysozyme (1%), were identified. Using bioinformatics, we identified a universal amyloid proteome signature, which has high sensitivity and specificity for amyloidosis similar to that of Congo red staining. We curated proteome databases which included variant proteins associated with systemic amyloidosis, and identified clonotypic immunoglobulin variable gene usage in immunoglobulin light chain amyloidosis, and the variant peptides in hereditary transthyretin amyloidosis. In conclusion, mass spectrometry-based proteomic analysis of subcutaneous fat aspirates offers a powerful tool for the diagnosis and typing of systemic amyloidosis. The assay reveals the underlying pathogenesis by identifying variable gene usage in immunoglobulin light chains and the variant peptides in hereditary amyloidosis. Copyright© Ferrata Storti Foundation.

Site specific modification of the human plasma proteome by methylglyoxal

International Nuclear Information System (INIS)

Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig S.; Monks, Terrence J.; Lau, Serrine S.

2015-01-01

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

Site specific modification of the human plasma proteome by methylglyoxal

Energy Technology Data Exchange (ETDEWEB)

Kimzey, Michael J.; Kinsky, Owen R. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Yassine, Hussein N. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Tsaprailis, George [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Stump, Craig S. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Southern Arizona VA Health Care System, Tucson, AZ 85723 (United States); Monks, Terrence J. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Lau, Serrine S., E-mail: lau@pharmacy.arizona.edu [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States)

2015-12-01

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

Proteomics in pulmonary research: selected methodical aspects

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Martin Petrek

2007-10-01

Full Text Available Recent years witness rapid expansion of applications of proteomics to clinical research including non-malignant lung disorders. These developments bring along the need for standardisation of proteomic experiments. This paper briefly reviews basic methodical aspects of appliedproteomic studies using SELDI-TOF mass spectrometry platform as example but also emphasizes general aspects of quality assurance in proteomics. Key-words: lung proteome, quality assurance, SELDI-TOF MS

The potato tuber mitochondrial proteome

DEFF Research Database (Denmark)

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...

Development of insusceptibility to serum factor during the radiation transformation process

International Nuclear Information System (INIS)

Terasima, T.; Yasukawa, M.; Kimura, M.

1983-01-01

The effects of using different assay sera on the X-ray-induced transformation yield in 10T1/2 mouse cells were studied. The use of newborn calf assay serum gave a 5 x greater value of transformation than foetal calf serum. This difference in transformation yield with different assay sera was used to investigate the time interval required for the development of insusceptibility of cells to serum factor. Sequential shifting from low yield sera (LYS) to high yield sera (HYS) showed that the radiation-initiated cells were most susceptible to HYS at the beginning of the assay culture but finally became insusceptible within 2 weeks of incubation. (U.K.)

Effects on pig immunophysiology, PBMC proteome and brain neurotransmitters caused by group mixing stress and human-animal relationship

Science.gov (United States)

Valent, Daniel; Arroyo, Laura; Peña, Raquel; Yu, Kuai; Carreras, Ricard; Mainau, Eva; Velarde, Antonio

2017-01-01

Peripheral blood mononuclear cells (PBMC) are an interesting sample for searching for biomarkers with proteomic techniques because they are easy to obtain and do not contain highly abundant, potentially masking proteins. Two groups of pigs (n = 56) were subjected to mixing under farm conditions and afterwards subjected to different management treatments: negative handling (NH) and positive handling (PH). Serum and PBMC samples were collected at the beginning of the experiment one week after mixing (t0) and after two months of different handling (t2). Brain areas were collected after slaughter and neurotransmitters quantified by HPLC. Hair cortisol and serum acute phase proteins decreased and serum glutathione peroxidase increased at t2, indicating a lower degree of stress at t2 after adaptation to the farm. Differential gel electrophoresis (DIGE) was applied to study the effects of time and treatment on the PBMC proteome. A total of 54 differentially expressed proteins were identified, which were involved in immune system modulation, cell adhesion and motility, gene expression, splicing and translation, protein degradation and folding, oxidative stress and metabolism. Thirty-seven protein spots were up-regulated at t2 versus t0 whereas 27 were down-regulated. Many of the identified proteins share the characteristic of being potentially up or down-regulated by cortisol, indicating that changes in protein abundance between t0 and t2 are, at least in part, consequence of lower stress upon adaptation to the farm conditions after group mixing. Only slight changes in brain neurotransmitters and PBMC oxidative stress markers were observed. In conclusion, the variation in hair cortisol and serum APPs as well as the careful analysis of the identified proteins indicate that changes in protein composition in PBMC throughout time is mainly due to a decrease in the stress status of the individuals, following accommodation to the farm and the new group. PMID:28475627

Effects on pig immunophysiology, PBMC proteome and brain neurotransmitters caused by group mixing stress and human-animal relationship.

Science.gov (United States)

Valent, Daniel; Arroyo, Laura; Peña, Raquel; Yu, Kuai; Carreras, Ricard; Mainau, Eva; Velarde, Antonio; Bassols, Anna

2017-01-01

Peripheral blood mononuclear cells (PBMC) are an interesting sample for searching for biomarkers with proteomic techniques because they are easy to obtain and do not contain highly abundant, potentially masking proteins. Two groups of pigs (n = 56) were subjected to mixing under farm conditions and afterwards subjected to different management treatments: negative handling (NH) and positive handling (PH). Serum and PBMC samples were collected at the beginning of the experiment one week after mixing (t0) and after two months of different handling (t2). Brain areas were collected after slaughter and neurotransmitters quantified by HPLC. Hair cortisol and serum acute phase proteins decreased and serum glutathione peroxidase increased at t2, indicating a lower degree of stress at t2 after adaptation to the farm. Differential gel electrophoresis (DIGE) was applied to study the effects of time and treatment on the PBMC proteome. A total of 54 differentially expressed proteins were identified, which were involved in immune system modulation, cell adhesion and motility, gene expression, splicing and translation, protein degradation and folding, oxidative stress and metabolism. Thirty-seven protein spots were up-regulated at t2 versus t0 whereas 27 were down-regulated. Many of the identified proteins share the characteristic of being potentially up or down-regulated by cortisol, indicating that changes in protein abundance between t0 and t2 are, at least in part, consequence of lower stress upon adaptation to the farm conditions after group mixing. Only slight changes in brain neurotransmitters and PBMC oxidative stress markers were observed. In conclusion, the variation in hair cortisol and serum APPs as well as the careful analysis of the identified proteins indicate that changes in protein composition in PBMC throughout time is mainly due to a decrease in the stress status of the individuals, following accommodation to the farm and the new group.

Label-free LC-MSe in tissue and serum reveals protein networks underlying differences between benign and malignant serous ovarian tumors.

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Wouter Wegdam

Full Text Available PURPOSE: To identify proteins and (molecular/biological pathways associated with differences between benign and malignant epithelial ovarian tumors. EXPERIMENTAL PROCEDURES: Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. RESULTS: In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. DISCUSSION: Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum

metabolicMine: an integrated genomics, genetics and proteomics data warehouse for common metabolic disease research.

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Lyne, Mike; Smith, Richard N; Lyne, Rachel; Aleksic, Jelena; Hu, Fengyuan; Kalderimis, Alex; Stepan, Radek; Micklem, Gos

2013-01-01

Common metabolic and endocrine diseases such as diabetes affect millions of people worldwide and have a major health impact, frequently leading to complications and mortality. In a search for better prevention and treatment, there is ongoing research into the underlying molecular and genetic bases of these complex human diseases, as well as into the links with risk factors such as obesity. Although an increasing number of relevant genomic and proteomic data sets have become available, the quantity and diversity of the data make their efficient exploitation challenging. Here, we present metabolicMine, a data warehouse with a specific focus on the genomics, genetics and proteomics of common metabolic diseases. Developed in collaboration with leading UK metabolic disease groups, metabolicMine integrates data sets from a range of experiments and model organisms alongside tools for exploring them. The current version brings together information covering genes, proteins, orthologues, interactions, gene expression, pathways, ontologies, diseases, genome-wide association studies and single nucleotide polymorphisms. Although the emphasis is on human data, key data sets from mouse and rat are included. These are complemented by interoperation with the RatMine rat genomics database, with a corresponding mouse version under development by the Mouse Genome Informatics (MGI) group. The web interface contains a number of features including keyword search, a library of Search Forms, the QueryBuilder and list analysis tools. This provides researchers with many different ways to analyse, view and flexibly export data. Programming interfaces and automatic code generation in several languages are supported, and many of the features of the web interface are available through web services. The combination of diverse data sets integrated with analysis tools and a powerful query system makes metabolicMine a valuable research resource. The web interface makes it accessible to first

Quantitative proteomic profiling of membrane proteins from the mouse brain cortex, hippocampus, and cerebellum using the HysTag reagent: mapping of neurotransmitter receptors and ion channels

DEFF Research Database (Denmark)

Olsen, Jesper V; Nielsen, Peter Aa; Andersen, Jens R

2007-01-01

of recently developed methods for isolation of membrane proteins from 10-20 mg brain tissue [Nielsen, P.Aa., Olsen, J.V., Podtelejnokov, A.V., Andersen, J.R., Mann, M., Wisniewski, J.R., 2005. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 4, 402--408] and the Hys...

Proteomic analysis of mouse thymoma EL4 cells treated with bis(tri-n-butyltin)oxide (TBTO).

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Osman, Ahmed M; van Kol, Sandra; Peijnenburg, Ad; Blokland, Marco; Pennings, Jeroen L A; Kleinjans, Jos C S; van Loveren, Henk

2009-09-01

Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the differentially expressed proteins with the corresponding mRNA expression data. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. The calculation of the ratio of peptides obtained from exposed and control samples allowed us to evaluate the effect of TBTO on protein expression and to compare these results to those obtained in gene expression profiling studies. Correlation of some of the differentially expressed proteins and their corresponding mRNAs was observed. The analysis of the protein ratios revealed that 12 proteins were significantly affected. These proteins included cytoskeleton proteins myosin-9, spectrin beta 2 and plectin 8. The first two proteins were down-regulated 3-fold, whereas the third was up-regulated 2-fold. Ras-related Rab1, a GTP binding protein and T-complex protein-1 subunit alpha, a chaperonin, were decreased 2- and 3.6-fold, respectively. The ribosomal S10 and eukaryotic translation factor (eIf4G1), which are involved in protein synthesis, were down-regulated 2.6- and 3.7-fold, respectively. Also, proteins involved in splicing of pre-mRNA and in transcription, splicing factor arginine/serine-rich 2 and chromodomain-helicase-DNA binding protein 4 (Chd4), were decreased 2.6- and 4.5 times, respectively. Nuclear RNA helicase II was reduced 2.8-fold. Finally, prothymosin-alpha (ProTalpha), an essential protein for cell proliferation, and a protein similar to ProTalpha, (with a molecular weight and a pI (3.54) comparable to that of ProTalpha) were also down-regulated 6-and 8-fold, respectively. We propose that the observed down-regulation of the expression

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

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Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

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Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

2010-01-03

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases. (c) 2009 Elsevier B.V. All rights reserved.

Multilayered Genetic and Omics Dissection of Mitochondrial Activity in a Mouse Reference Population

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Wu, Yibo; Williams, Evan G.; Dubuis, Sébastien; Mottis, Adrienne; Jovaisaite, Virginija; Houten, Sander M.; Argmann, Carmen A.; Faridi, Pouya; Wolski, Witold; Kutalik, Zoltán; Zamboni, Nicola; Auwerx, Johan; Aebersold, Ruedi

2014-01-01

SUMMARY The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome—a subset of the metabolome—and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPRmt). UPRmt shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes. PMID:25215496

Proteomic Analysis of Chinese Hamster Ovary Cells

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Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama

2012-01-01

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

Semen proteomics and male infertility.

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Jodar, Meritxell; Soler-Ventura, Ada; Oliva, Rafael

2017-06-06

Semen is a complex body fluid containing an admixture of spermatozoa suspended in secretions from the testes and epididymis which are mixed at the time of ejaculation with secretions from other accessory sex glands such as the prostate and seminal vesicles. High-throughput technologies have revealed that, contrary to the idea that sperm cells are simply a silent delivery vehicle of the male genome to the oocyte, the sperm cells in fact provide both a specific epigenetically marked DNA together with a complex population of proteins and RNAs crucial for embryogenesis. Similarly, -omic technologies have also enlightened that seminal fluid seems to play a much greater role than simply being a medium to carry the spermatozoa through the female reproductive tract. In the present review, we briefly overview the sperm cell biology, consider the key issues in sperm and seminal fluid sample preparation for high-throughput proteomic studies, describe the current state of the sperm and seminal fluid proteomes generated by high-throughput proteomic technologies and provide new insights into the potential communication between sperm and seminal fluid. In addition, comparative proteomic studies open a window to explore the potential pathogenic mechanisms of infertility and the discovery of potential biomarkers with clinical significance. The review updates the numerous proteomics studies performed on semen, including spermatozoa and seminal fluid. In addition, an integrative analysis of the testes, sperm and seminal fluid proteomes is also included providing insights into the molecular mechanisms that regulate the generation, maturation and transit of spermatozoa. Furthermore, the compilation of several differential proteomic studies focused on male infertility reveals potential pathways disturbed in specific subtypes of male infertility and points out towards future research directions in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project.

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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.

Increased serum erythropoietin activity in rats following intrarenal injection of nickel subsulfide

International Nuclear Information System (INIS)

Hopfer, S.M.; Sunderman, F.W. Jr.; Fredrickson, T.N.; Morse, E.E.

1979-01-01

To investigate the pathopysiologic mechanisms of nickel-induced erythocytosis, serum erythropoietin activities were measured in (a) pooled serum from rats at 2 wk after intrarenal injection of αNi 3 S 2 (5 mg/rat), and (b) pooled serum from control rats at 2 wk after intrarenal injection of sterile NaCl vehicle (0.4 ml/rat). A sensitive erythropoietin bioassay was employed, which entailed repetitive administration of test serums to post-hypoxic polycythemic mice in divided doses (12 s.c. injections of 0.5 ml of serum at 6 h intervals for 3 da; total dose = 6 ml of serum/mouse). The erythropoietin detection limit was approx. = 20 I.U./liter of serum. In mice which received pooled serum from αNi 3 S 2 -treated rats, erythrocyte 59 Fe-uptake averaged 28% (S.D. +- 5) (vs 3.7 +- 1.1% in control rats; P 3 S 2 -treated rats averaged 130 I.U./liter (S.D. +- 18) (vs 27 +- 6 I.U./liter in control rats; P 3 S 2 is mediated by increased serum erythropoietin activity

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography-MS/MS analysis: Application to a pharmacokinetic study.

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Szczesny, Damian; Bartosińska, Ewa; Jacyna, Julia; Patejko, Małgorzata; Siluk, Danuta; Kaliszan, Roman

2018-02-01

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI-MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile-ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed. Copyright © 2017 John Wiley & Sons, Ltd.

Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

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Brian A Chow

Full Text Available Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

Proteomic approaches in brain research and neuropharmacology.

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Vercauteren, Freya G G; Bergeron, John J M; Vandesande, Frans; Arckens, Lut; Quirion, Rémi

2004-10-01

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznan, Poland

Czech Academy of Sciences Publication Activity Database

Gadher, S. J.; Marczak, L.; Luczak, M.; Stobiecki, M.; Widlak, P.; Kovářová, Hana

2016-01-01

Roč. 13, č. 1 (2016), s. 5-7 ISSN 1478-9450. [Central and Eastern European Proteomic Conference (CEEPC) /9./. Poznaň, 15.06.2015-18.06.2015] Institutional support: RVO:67985904 Keywords : Central and Eastern Proteomic Conference * proteomics * mass spectrometry imaging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.849, year: 2016

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant

NARCIS (Netherlands)

Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A.; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B.; Hettinga, Kasper

2016-01-01

Here we provide data from shot-gun proteomics, using filtered-aided sample preparation (FASP), dimethyl labeling and LC-MS/MS, to quantify the changes in the repertoire of human milk proteins over lactation. Milk serum proteins were analyzed at week 1, 2, 3 4, 8, 16, and 24 in milk from four

Dynamic adaptation of myocardial proteome during heart failure development

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Poesch, Axel; Dörr, Marcus; Völker, Uwe; Grube, Karina; Hammer, Elke; Felix, Stephan B.

2017-01-01

Heart failure (HF) development is characterized by huge structural changes that are crucial for disease progression. Analysis of time dependent global proteomic adaptations during HF progression offers the potential to gain deeper insights in the disease development and identify new biomarker candidates. Therefore, hearts of TAC (transverse aortic constriction) and sham mice were examined by cardiac MRI on either day 4, 14, 21, 28, 42, and 56 after surgery (n = 6 per group/time point). At each time point, proteomes of the left (LV) and right ventricles (RV) of TAC and sham mice were analyzed by mass spectrometry (MS). In TAC mice, systolic LV heart function worsened from day 4 to day 14, remained on a stable level from day 14 to day 42, and showed a further pronounced decline at day 56. MS analysis identified in the LV 330 and in RV 246 proteins with altered abundance over time (TAC vs. sham, fc≥±2). Functional categorization of proteins disclosed the time-dependent alteration of different pathways. Heat shock protein beta-7 (HSPB7) displayed differences in abundance in tissue and serum at an early stage of HF. This study not only provides an overview of the time dependent molecular alterations during transition to HF, but also identified HSPB7 as a novel blood biomarker candidate for the onset of cardiac remodeling. PMID:28973020

Establishing Substantial Equivalence: Proteomics

Science.gov (United States)

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.

Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme

International Nuclear Information System (INIS)

Saxe, A.W.; Hollinger, M.A.; Essam, T.

1986-01-01

The effect of bilirubin on serum angiotensin-converting enzyme (ACE) activity was studied with spectrophotometric and radionuclide assays. In the spectrophotometric assay addition of bilirubin to normal serum from dog, mouse, and human produced a dose-related inhibition of ACE activity. A 50% decrease in human ACE activity was produced by the addition of approximately 250 mg/L in vitro. Serum from icteric patients with elevated bilirubin was also associated with a reduction in ACE activity in the spectrophotometric assay. A 50% decrease in ACE activity in these samples was associated with a serum bilirubin of approximately 220 mg/L. In the radionuclide assay, however, addition of bilirubin to normal human serum failed to reduce measured ACE activity. The use of a radionuclide assay for serum ACE in clinical samples offers the advantage of less interference from serum bilirubin

Identification of Biomarkers for Endometriosis Using Clinical Proteomics

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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.

Proteome identification of the silkworm middle silk gland

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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

Probing cocaine-antibody interactions in buffer and human serum.

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Muthu Ramakrishnan

Full Text Available Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST, isothermal titration calorimetry (ITC, and surface plasmon resonance (SPR we have evaluated the affinity properties of a representative mouse monoclonal (mAb08 as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum.MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC. This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies.High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide

Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

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Sandra Murphy

2015-06-01

Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

Science.gov (United States)

Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

2018-03-19

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to

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

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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.

Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

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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.

Application of Fluorescence Two-Dimensional Difference In-Gel Electrophoresis as a Proteomic Biomarker Discovery Tool in Muscular Dystrophy Research

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Carberry, Steven; Zweyer, Margit; Swandulla, Dieter; Ohlendieck, Kay

2013-01-01

In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue. PMID:24833232

Comparison of protein extraction methods suitable for proteomics ...

African Journals Online (AJOL)

Jane

2011-07-27

Jul 27, 2011 ... An efficient protein extraction method is a prerequisite for successful implementation of proteomics. ... research, it is noteworthy to discover a proteome ..... Proteomic analysis of rice (Oryza sativa) seeds during germination.

Plasma proteomic study in patients with high altitude pulmonary edema (HAPE

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Yong-jun LUO

2012-01-01

Full Text Available Objective  To investigate the differential expressions of protein in the plasma proteome in patients suffering from high altitude pulmonary edema (HAPE and their implications. Methods  The plasmas of six HAPE patients and six healthy controls were studied. The high-abundant proteins in the plasma were removed. The low-abundant proteins in the plasma/serum were segregated by 2-DE. MALDI-TOF/MS was adopted to measure the peptide fingerprints after the differential protein spots were digested by enzymes. Comparison and analysis were made in the GenBank. Results  The immunoglobulin K1 light chain, serum transferrin protein precursor, and α-trypsin inhibitor heavy chain-related protein expressions were upregulated in HAPE patients compared with the control group. However the human fibrin glue coagulation protein 3 was down-regulated. Conclusion  The differential expression of the above four proteins in the plasma of HAPE patients may be related to the occurrence of HAPE and can be used as the target point for the prediction of HAPE.

Urine Proteomics in the Era of Mass Spectrometry

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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.

Proteomics of Plant Pathogenic Fungi

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Raquel González-Fernández

2010-01-01

Full Text Available Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

Birth of plant proteomics in India: a new horizon.

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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.

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

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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.

The Redox Proteome*

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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

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznań, Poland.

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Gadher, Suresh Jivan; Marczak, Łukasz; Łuczak, Magdalena; Stobiecki, Maciej; Widlak, Piotr; Kovarova, Hana

2016-01-01

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznań, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznań.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

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José B Gama

2014-08-01

Full Text Available Buruli ulcer (BU is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1 and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1. In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

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Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

2014-08-01

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Application of proteomics to ecology and population biology.

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Karr, T L

2008-02-01

Proteomics is a relatively new scientific discipline that merges protein biochemistry, genome biology and bioinformatics to determine the spatial and temporal expression of proteins in cells, tissues and whole organisms. There has been very little application of proteomics to the fields of behavioral genetics, evolution, ecology and population dynamics, and has only recently been effectively applied to the closely allied fields of molecular evolution and genetics. However, there exists considerable potential for proteomics to impact in areas related to functional ecology; this review will introduce the general concepts and methodologies that define the field of proteomics and compare and contrast the advantages and disadvantages with other methods. Examples of how proteomics can aid, complement and indeed extend the study of functional ecology will be discussed including the main tool of ecological studies, population genetics with an emphasis on metapopulation structure analysis. Because proteomic analyses provide a direct measure of gene expression, it obviates some of the limitations associated with other genomic approaches, such as microarray and EST analyses. Likewise, in conjunction with associated bioinformatics and molecular evolutionary tools, proteomics can provide the foundation of a systems-level integration approach that can enhance ecological studies. It can be envisioned that proteomics will provide important new information on issues specific to metapopulation biology and adaptive processes in nature. A specific example of the application of proteomics to sperm ageing is provided to illustrate the potential utility of the approach.

Virtual Labs in proteomics: new E-learning tools.

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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.

Moesin Is a Biomarker for the Assessment of Genotoxic Carcinogens in Mouse Lymphoma

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

2012-01-01

1,2-Dibromoethane and glycidol are well known genotoxic carcinogens, which have been widely used in industry. To identify a specific biomarker for these carcinogens in cells, the cellular proteome of L5178Y mouse lymphoma cells treated with these compounds was analyzed by 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS). Of 50 protein spots showing a greater than 1.5-fold increase or decrease in intensity compared to control cells on a 2-D gel, we focused on the candidate biomarker moesin. Western analysis using monoclonal rabbit anti-moesin confirmed the identity of the protein and its increased level of expression upon exposure to the carcinogenic compounds. Moesin expression also increased in cells treated with six additional genotoxic carcinogens, verifying that moesin could serve as a biomarker to monitor phenotypic change upon exposure to genotoxic carcinogens in L5178Y mouse lymphoma cells. PMID:22358511

Analysis of mass spectrometry data in proteomics

DEFF Research Database (Denmark)

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....

Maillard Proteomics: Opening New Pages

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Alena Soboleva

2017-12-01

Full Text Available Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus, proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

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Rita E. Serda

2011-01-01

Full Text Available Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

Science.gov (United States)

Serda, Rita E.; Blanco, Elvin; Mack, Aaron; Stafford, Susan J.; Amra, Sarah; Li, Qingpo; van de Ven, Anne L.; Tanaka, Takemi; Torchilin, Vladimir P.; Wiktorowicz, John E.; Ferrari, Mauro

2014-01-01

Mass transport of drug delivery vehicles is guided by particle properties, such as shape, composition and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light chain variable region, fibrinogen, and complement component 1 compared to their anionic counterparts. The anionic-surface favored equal accumulation of microparticles in the liver and spleen, while cationic-surfaces favored preferential accumulation in the spleen. Immunohistochemistry supported macrophage internalization of both anionic and cationic silicon microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution. PMID:21303614

Knowledge Translation: Moving Proteomics Science to Innovation in Society.

Science.gov (United States)

Holmes, Christina; McDonald, Fiona; Jones, Mavis; Graham, Janice

2016-06-01

Proteomics is one of the pivotal next-generation biotechnologies in the current "postgenomics" era. Little is known about the ways in which innovative proteomics science is navigating the complex socio-political space between laboratory and society. It cannot be assumed that the trajectory between proteomics laboratory and society is linear and unidirectional. Concerned about public accountability and hopes for knowledge-based innovations, funding agencies and citizens increasingly expect that emerging science and technologies, such as proteomics, are effectively translated and disseminated as innovation in society. Here, we describe translation strategies promoted in the knowledge translation (KT) and science communication literatures and examine the use of these strategies within the field of proteomics. Drawing on data generated from qualitative interviews with proteomics scientists and ethnographic observation of international proteomics conferences over a 5-year period, we found that proteomics science incorporates a variety of KT strategies to reach knowledge users outside the field. To attain the full benefit of KT, however, proteomics scientists must challenge their own normative assumptions and approaches to innovation dissemination-beyond the current paradigm relying primarily on publication for one's scientific peers within one's field-and embrace the value of broader (interdisciplinary) KT strategies in promoting the uptake of their research. Notably, the Human Proteome Organization (HUPO) is paying increasing attention to a broader range of KT strategies, including targeted dissemination, integrated KT, and public outreach. We suggest that increasing the variety of KT strategies employed by proteomics scientists is timely and would serve well the omics system sciences community.

[Detection of fps tumor antigen with mono-specific anti-fps serum in tumors induced by acute transforming ALV].

Science.gov (United States)

Wang, Yixin; Chen, Hao; Zhao, Peng; Li, Jianliang; Cui, Zhizhong

2013-03-04

To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.

First systematic plant proteomics workshop in Botany Department, University of Delhi: transferring proteomics knowledge to next-generation researchers and students.

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Deswal, Renu; Abat, Jasmeet Kaur; Sehrawat, Ankita; Gupta, Ravi; Kashyap, Prakriti; Sharma, Shruti; Sharma, Bhavana; Chaurasia, Satya Prakash; Chanu, Sougrakpam Yaiphabi; Masi, Antonio; Agrawal, Ganesh Kumar; Sarkar, Abhijit; Agrawal, Raj; Dunn, Michael J; Renaut, Jenny; Rakwal, Randeep

2014-07-01

International Plant Proteomics Organization (INPPO) outlined ten initiatives to promote plant proteomics in each and every country. With greater emphasis in developing countries, one of those was to "organize workshops at national and international levels to train manpower and exchange information". This third INPPO highlights covers the workshop organized for the very first time in a developing country, India, at the Department of Botany in University of Delhi on December 26-30, 2013 titled - "1(st) Plant Proteomics Workshop / Training Program" under the umbrella of INPPO India-Nepal chapter. Selected 20 participants received on-hand training mainly on gel-based proteomics approach along with manual booklet and parallel lectures on this and associated topics. In house, as well as invited experts drawn from other Universities and Institutes (national and international), delivered talks on different aspects of gel-based and gel-free proteomics. Importance of gel-free proteomics approach, translational proteomics, and INPPO roles were presented and interactively discussed by a group of three invited speakers Drs. Ganesh Kumar Agrawal (Nepal), Randeep Rakwal (Japan), and Antonio Masi (Italy). Given the output of this systematic workshop, it was proposed and thereafter decided to be organized every alternate year; the next workshop will be held in 2015. Furthermore, possibilities on providing advanced training to those students / researchers / teachers with basic knowledge in proteomics theory and experiments at national and international levels were discussed. INPPO is committed to generating next-generation trained manpower in proteomics, and it would only happen by the firm determination of scientists to come forward and do it. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Systems biology integration of proteomic data in rodent models of depression reveals involvement of the immune response and glutamatergic signaling.

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Carboni, Lucia; Nguyen, Thanh-Phuong; Caberlotto, Laura

2016-12-01

The pathophysiological basis of major depression is incompletely understood. Recently, numerous proteomic studies have been performed in rodent models of depression to investigate the molecular underpinnings of depressive-like behaviours with an unbiased approach. The objective of the study is to integrate the results of these proteomic studies in depression models to shed light on the most relevant molecular pathways involved in the disease. Network analysis is performed integrating preexisting proteomic data from rodent models of depression. The IntAct mouse and the HRPD are used as reference protein-protein interaction databases. The functionality analyses of the networks are then performed by testing overrepresented GO biological process terms and pathways. Functional enrichment analyses of the networks revealed an association with molecular processes related to depression in humans, such as those involved in the immune response. Pathways impacted by clinically effective antidepressants are modulated, including glutamatergic signaling and neurotrophic responses. Moreover, dysregulations of proteins regulating energy metabolism and circadian rhythms are implicated. The comparison with protein pathways modulated in depressive patients revealed significant overlapping. This systems biology study supports the notion that animal models can contribute to the research into the biology and therapeutics of depression. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of proteomic biomarker panels in urine and serum for ovarian cancer diagnosis

DEFF Research Database (Denmark)

Petri, Anette Lykke; Simonsen, Anja Hviid; Høgdall, Estrid

2010-01-01

The purposes of this study were to confirm previously found candidate epithelial ovarian cancer biomarkers in urine and to compare a paired serum biomarker panel and a urine biomarker panel from the same study cohort with regard to the receiver operating characteristic curve (ROC) area under the ...

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum

DEFF Research Database (Denmark)

Banda, Nirmal K; Mehta, Gaurav; Chao, Ying

2014-01-01

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has...... the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via...... the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human...

Proteomics methods applied to malaria: Plasmodium falciparum

International Nuclear Information System (INIS)

Cuesta Astroz, Yesid; Segura Latorre, Cesar

2012-01-01

Malaria is a parasitic disease that has a high impact on public health in developing countries. The sequencing of the plasmodium falciparum genome and the development of proteomics have enabled a breakthrough in understanding the biology of the parasite. Proteomics have allowed to characterize qualitatively and quantitatively the parasite s expression of proteins and has provided information on protein expression under conditions of stress induced by antimalarial. Given the complexity of their life cycle, this takes place in the vertebrate host and mosquito vector. It has proven difficult to characterize the protein expression during each stage throughout the infection process in order to determine the proteome that mediates several metabolic, physiological and energetic processes. Two dimensional electrophoresis, liquid chromatography and mass spectrometry have been useful to assess the effects of antimalarial on parasite protein expression and to characterize the proteomic profile of different p. falciparum stages and organelles. The purpose of this review is to present state of the art tools and advances in proteomics applied to the study of malaria, and to present different experimental strategies used to study the parasite's proteome in order to show the advantages and disadvantages of each one.

Protective effect of Bifidobacterium infantis CGMCC313-2 on ovalbumin-induced airway asthma and β-lactoglobulin-induced intestinal food allergy mouse models

Science.gov (United States)

Liu, Meng-Yun; Yang, Zhen-Yu; Dai, Wen-Kui; Huang, Jian-Qiong; Li, Yin-Hu; Zhang, Juan; Qiu, Chuang-Zhao; Wei, Chun; Zhou, Qian; Sun, Xin; Feng, Xin; Li, Dong-Fang; Wang, He-Ping; Zheng, Yue-Jie

2017-01-01

AIM To determine whether oral administration of Bifidobacterium infantis CGMCC313-2 (B. infantis CGMCC313-2) inhibits allergen-induced airway inflammation and food allergies in a mouse model. METHODS Ovalbumin (OVA)-induced allergic asthma and β-lactoglobulin-induced food allergy mouse models were used in this study. Following oral administration of B. infantis CGMCC313-2 during or after allergen sensitization, histopathologic changes in the lung and intestine were evaluated by hematoxylin and eosin (HE) staining. In the allergic asthma mouse model, we evaluated the proportion of lung-infiltrating inflammatory cells. OVA-specific IgE and IgG1 levels in serum and cytokine levels in bronchoalveolar lavage fluid (BALF) were also assessed. In the food allergy mouse model, the levels of total IgE and cytokines in serum were measured. RESULTS Oral administration of B. infantis CGMCC313-2 during or after allergen sensitization suppressed allergic inflammation in lung and intestinal tissues, while the proportion of infiltrating inflammatory cells was significantly decreased in the BALF of allergic asthma mice. Moreover, B. infantis CGMCC313-2 decreased the serum levels of total IgE in food allergy mice, and reductions in IgE and IgG1 were also observed in OVA-induced allergic asthma mice. The expression of interleukin-4 (IL-4) and IL-13 in both serum and BALF was suppressed following the administration of B. infantis CGMCC313-2, while an effect on serum IL-10 levels was not observed. CONCLUSION B. infantis CGMCC313-2 inhibits the secretion of allergen-induced IgE, IL-4 and IL-13, and attenuates allergic inflammation. PMID:28405142

MS-based monitoring of proteolytic decay of synthetic reporter peptides for quality control of plasma and serum specimens.

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Findeisen, Peter; Thumfart, Jörg Oliver; Costina, Victor; Hofheinz, Ralf; Neumaier, Michael

2013-09-01

To determine the preanalytical quality of serum and plasma by monitoring the time-dependent ex vivo decay of a synthetic reporter peptide (RP) with liquid chromatography/mass spectrometry (LC/MS). Serum and plasma specimens were spiked with the RP and proteolytic fragments were monitored with LC/MS at different preanalytical time points ranging from 2 to 24 hours after blood withdrawal. The concentration of fragments changed in a time-dependent manner, and respective peptide profiles were used to classify specimens according to their preanalytical time span. Classification accuracy was high, with values always above 0.89 for areas under receiver operating characteristic curves. This "proteomics degradation clock" can be used to estimate the preanalytical quality of serum and plasma and might have impact on quality control procedures of biobanking repositories.

Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis Tratamento de soro com sobrenadante de cultura de Candida albicans reduz a fagocitose soro-dependente

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Aderbal Antonio dos Santos

2002-01-01

Full Text Available Candida albicans is a potent activator of the complement system, and heat labile opsonins produced by activation of C3 (C3b and iC3b enhance phagocytosis of C. albicans mediated by complement receptors. In this study we treated mouse serum with supernatants from cultures of a protease producer strain of C. albicans and evaluated the ability of this serum to enhance phagocytosis of C. albicans. Cell-free supernatants from cultures of C. albicans were concentrated 5 fold and added to mouse serum for 30 min at 37ºC, before using this serum for opsonization of glutaraldehyde-fixed yeast cells. We observed that normal mouse serum increased about 3 fold the phagocytosis of C. albicans by mice peritoneal macrophages, whereas supernatant-treated serum did not increase phagocytosis. This effect of supernatants on serum was prevented by addition of pepstatin (5 µg/ ml; an inhibitor of C. albicans acid proteases to the medium. Serum treated with supernatants from cultures of a protease-deficient mutant of C. albicans also increased about 3 fold phagocytosis of the yeast. These results suggest that a protease produced by C. albicans causes proteolysis of serum opsonins, thereby reducing the phagocytosis of the yeast.Candida albicans é um potente ativador do sistema complemento, e opsoninas lábeis ao calor produzidas por ativação de C3 (C3b e iC3b aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de células obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37ºC, antes deste soro ser usado para opsonização de C. albicans na forma de levedura e fixadas em glutaraldeido. Nós observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por

Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1R-(-)-menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

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Zhao, Jing; Jin, Yan; Shin, Yujin; Jeong, Kyung Min; Lee, Jeongmi

2016-03-01

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 μL of serum, followed by derivatization of the extracts in 50 μL of 2 mM 1R-(-)-menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub-2 μm C18 column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0-1000.0 ng/mL for both R and S enantiomers (r(2) > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R- and S-fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Seed Proteome Web Portal

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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.

γ-Oryzanol recovers mouse hypoadiponectinemia induced by animal fat ingestion.

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Nagasaka, Reiko; Yamsaki, Tomoteru; Uchida, Asako; Ohara, Kazuyuki; Ushio, Hideki

2011-06-15

Adiponectin is an insulin-sensitizing adipocyte-derived adipokine. The decrease in plasma adiponectin level (hypoadiponectinemia) is involved in the development of insulin resistance and the resulting type 2 diabetes. Our previous studies have demonstrated that γ-oryzanol (ORZ) from rice bran suppressed NF-κB activation and increased adiponectin secretion from adipocyte. In this study, we have evaluated effects of oral administration of animal fat (beef tallow) and palmitate on mouse serum adiponectin level. Oral administrations of beef tallow and palmitate significantly suppressed serum adiponectin levels into around half of the initial level from 48 to 96 h after administration compared with the case of corn oil (P<0.05). Coadministration of ORZ successfully remedied mouse hypoadiponectinemia induced by ingestion of beef tallow and the relative adiponectin levels attained to 1.66±0.23 at 96 h after administration (mean value±s.e., P<0.05). Diverse physiological functions of ORZ in crop bran might be promising us to prevent chronic inflammations in the pathogeneses of the metabolic or insulin resistance syndromes. Copyright © 2011 Elsevier GmbH. All rights reserved.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

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Zhou, Yihua; Xu, Bixiong C.; Maheshwari, Hiralal G.; He, Li; Reed, Michael; Lozykowski, Maria; Okada, Shigeru; Cataldo, Lori; Coschigamo, Karen; Wagner, Thomas E.; Baumann, Gerhard; Kopchick, John J.

1997-01-01

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans. PMID:9371826

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

Science.gov (United States)

Zhou, Y; Xu, B C; Maheshwari, H G; He, L; Reed, M; Lozykowski, M; Okada, S; Cataldo, L; Coschigamo, K; Wagner, T E; Baumann, G; Kopchick, J J

1997-11-25

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Molecular stratification and precision medicine in systemic sclerosis from genomic and proteomic data.

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Martyanov, Viktor; Whitfield, Michael L

2016-01-01

The goal of this review is to summarize recent advances into the pathogenesis and treatment of systemic sclerosis (SSc) from genomic and proteomic studies. Intrinsic gene expression-driven molecular subtypes of SSc are reproducible across three independent datasets. These subsets are a consistent feature of SSc and are found in multiple end-target tissues, such as skin and esophagus. Intrinsic subsets as well as baseline levels of molecular target pathways are potentially predictive of clinical response to specific therapeutics, based on three recent clinical trials. A gene expression-based biomarker of modified Rodnan skin score, a measure of SSc skin severity, can be used as a surrogate outcome metric and has been validated in a recent trial. Proteome analyses have identified novel biomarkers of SSc that correlate with SSc clinical phenotypes. Integrating intrinsic gene expression subset data, baseline molecular pathway information, and serum biomarkers along with surrogate measures of modified Rodnan skin score provides molecular context in SSc clinical trials. With validation, these approaches could be used to match patients with the therapies from which they are most likely to benefit and thus increase the likelihood of clinical improvement.

Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse.

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Winding, Christiana; Sun, Yanwei; Höger, Harald; Bubna-Littitz, Hermann; Pollak, Arnold; Schmidt, Peter; Lubec, Gert

2011-06-01

Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Liver proteomics in progressive alcoholic steatosis

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Fernando, Harshica [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Wiktorowicz, John E.; Soman, Kizhake V. [Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Kaphalia, Bhupendra S.; Khan, M. Firoze [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Shakeel Ansari, G.A., E-mail: sansari@utmb.edu [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

2013-02-01

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3

Metabolic profiles in serum of mouse after chronic exposure to drinking water.

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Zhang, Yan; Wu, Bing; Zhang, Xuxiang; Li, Aimin; Cheng, Shupei

2011-08-01

The toxicity of Nanjing drinking water on mouse (Mus musculus) was detected by (1)H nuclear magnetic resonance (NMR)-based metabonomic method. Three groups of mice were fed with drinking water (produced by Nanjing BHK Water Plant), 3.8 μg/L benzo(a)pyrene as contrast, and clean water as control, respectively, for 90 days. It was observed that the levels of lactate, alanine, and creatinine in the mice fed with drinking water were increased and that of valine was decreased. The mice of drinking water group were successfully separated from control. The total concentrations of polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), and other organic pollutants in the drinking water were 0.23 μg/L, 4.57 μg/L, and 0.34 μg/L, respectively. In this study, Nanjing drinking water was found to induce distinct perturbations of metabolic profiles on mouse including disorders of glucose-alanine cycle, branched-chain amino acid and energy metabolism, and dysfunction of kidney. This study suggests that metabonomic method is feasible and sensitive to evaluate potential toxic effects of drinking water.

Dentistry proteomics: from laboratory development to clinical practice.

Science.gov (United States)

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.

Lens proteome map and alpha-crystallin profile of the catfish Rita rita.

Science.gov (United States)

Mohanty, Bimal Prasanna; Bhattacharjee, Soma; Das, Manas Kumar

2011-02-01

Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. alphaA-crystallins, member of the alpha-crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1- and 2-D immunoblot analysis with anti-alphaA-crystallin antibody. Two protein bands of 19-20 kDa were identified as alphaA-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-alphaB-crystallin and antiphospho-alphaB-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating alphaB-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita alpha-crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its alphaA- and alphaB-crystallin content (contain low amount from 5-9% of alphaB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse alpha-crystallins (contain higher amount of alphaB-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the alpha-crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of alphaA- and alphaB-isoforms, as

Marine proteomics: a critical assessment of an emerging technology.

Science.gov (United States)

Slattery, Marc; Ankisetty, Sridevi; Corrales, Jone; Marsh-Hunkin, K Erica; Gochfeld, Deborah J; Willett, Kristine L; Rimoldi, John M

2012-10-26

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Analysis of Serum Proteom after Intravenous Injection of cultivated wild ginseng pharmacopuncture

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Dong-Hee,Lee

2006-06-01

Full Text Available Objectives : To observe the changes in the serum proteins after intravenous injection of cultivated wild ginseng pharmacopuncture. Methods : Blood was collected before and after the administration of cultivated wild ginseng pharmacopuncture and only the serum was taken. Then differences in the spots on the scanned image after carrying out 2-Dimensional electrophoresis were located and conducted mass analysis and protein identification. Results : Following results were obtained from the comparative analysis of serum proteins before and after the administration of cultivated wild ginseng pharmacopuncture. 1. 28 spots were identified before and after the administration. 2. In confirming manifestation degree, spots with more than two-times increase were 204, 1302, 2205, 3105, 7104, 8006, spots with more than one-time increase were 1101, 1505, 2013, 2403, 3009, 3010, 4002, 4009, 6704, 8101, and spots with decrease were 205, 801, 803, 3205, 5202, 6105, 6106, 7103, 9001, 9003. 3. After conducting protein identification, proteins 205, 804, 1302, 4009, 6105, 6106 are unidentified yet, and 1l01 is unnamed protein. Protein 204 is identified as complement receptor CR2-C3d, 801 as YAPl protein, 803 as antitrypsin polymer, 1505 as PRO0684, 2013 and 3010 as proapolipoprotein, 2205 as USP48, 2403 as vitamin D binding protein, 3009 as complement component 4A preprotein, 3105 as immunoglobulin lambda chain, 3205 as transthyretin, 4002 as Ras-related protein Ral-A, 4204 as beta actin, 5202 and 7104 as apolipoprotein Ll, 6704 as alpha 2 macroglobulin precursor, 7103 as complement component 3 precursor, 8006 as testis-specific protein Y, 8101 as transferrin, 9001 as (Alpha-Oxy, Beta-(Cl12gdeoxy T-State Human Hemoglobin, and 9003 as human hemoglobin. 4. Immune protein CR2-C3d(204, which acts against microbes and pathogenic organisms, was increased by more than two-times after the administration of pharmacopuncture. 5. Antitrypsin(803, which is secreted with

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

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Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Proteogenomics Dashboard for the Human Proteome Project.

Science.gov (United States)

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.

Proteomic Biomarkers for Spontaneous Preterm Birth

DEFF Research Database (Denmark)

Kacerovsky, Marian; Lenco, Juraj; Musilova, Ivana

2014-01-01

This review aimed to identify, synthesize, and analyze the findings of studies on proteomic biomarkers for spontaneous preterm birth (PTB). Three electronic databases (Medline, Embase, and Scopus) were searched for studies in any language reporting the use of proteomic biomarkers for PTB published...

Plasma Proteome Biomarkers of Inflammation in School Aged Children in Nepal.

Directory of Open Access Journals (Sweden)

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

A Selected Reaction Monitoring Mass Spectrometry Protocol for Validation of Proteomic Biomarker Candidates in Studies of Psychiatric Disorders.

Science.gov (United States)

Reis-de-Oliveira, Guilherme; Garcia, Sheila; Guest, Paul C; Cassoli, Juliana S; Martins-de-Souza, Daniel

2017-01-01

Most biomarker candidates arising from proteomic studies of psychiatric disorders have not progressed for use in clinical studies due to insufficient validation steps. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS) approach that could be used as a follow-up validation tool of proteins identified in blood serum or plasma. This protocol specifically covers the stages of peptide selection and optimization. The increasing application of SRM-MS should enable fast, sensitive, and robust methods with the potential for use in clinical studies involving sampling of serum or plasma. Understanding the molecular mechanisms and identifying potential biomarkers for risk assessment, diagnosis, prognosis, and prediction of drug response goes toward the implementation of translational medicine strategies for improved treatment of patients with psychiatric disorders and other debilitating diseases.

Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

Science.gov (United States)

DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

2016-01-01

Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

Science.gov (United States)

Tikka, Saara; Monogioudi, Evanthia; Gotsopoulos, Athanasios; Soliymani, Rabah; Pezzini, Francesco; Scifo, Enzo; Uusi-Rauva, Kristiina; Tyynelä, Jaana; Baumann, Marc; Jalanko, Anu; Simonati, Alessandro; Lalowski, Maciej

2016-03-01

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood

Improving HIV proteome annotation: new features of BioAfrica HIV Proteomics Resource.

Science.gov (United States)

Druce, Megan; Hulo, Chantal; Masson, Patrick; Sommer, Paula; Xenarios, Ioannis; Le Mercier, Philippe; De Oliveira, Tulio

2016-01-01

The Human Immunodeficiency Virus (HIV) is one of the pathogens that cause the greatest global concern, with approximately 35 million people currently infected with HIV. Extensive HIV research has been performed, generating a large amount of HIV and host genomic data. However, no effective vaccine that protects the host from HIV infection is available and HIV is still spreading at an alarming rate, despite effective antiretroviral (ARV) treatment. In order to develop effective therapies, we need to expand our knowledge of the interaction between HIV and host proteins. In contrast to virus proteins, which often rapidly evolve drug resistance mutations, the host proteins are essentially invariant within all humans. Thus, if we can identify the host proteins needed for virus replication, such as those involved in transporting viral proteins to the cell surface, we have a chance of interrupting viral replication. There is no proteome resource that summarizes this interaction, making research on this subject a difficult enterprise. In order to fill this gap in knowledge, we curated a resource presents detailed annotation on the interaction between the HIV proteome and host proteins. Our resource was produced in collaboration with ViralZone and used manual curation techniques developed by UniProtKB/Swiss-Prot. Our new website also used previous annotations of the BioAfrica HIV-1 Proteome Resource, which has been accessed by approximately 10 000 unique users a year since its inception in 2005. The novel features include a dedicated new page for each HIV protein, a graphic display of its function and a section on its interaction with host proteins. Our new webpages also add information on the genomic location of each HIV protein and the position of ARV drug resistance mutations. Our improved BioAfrica HIV-1 Proteome Resource fills a gap in the current knowledge of biocuration.Database URL:http://www.bioafrica.net/proteomics/HIVproteome.html. © The Author(s) 2016. Published

Proteomics and the dynamic plasma membrane

DEFF Research Database (Denmark)

Sprenger, Richard R; Jensen, Ole Nørregaard

2010-01-01

plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

PROTEOMICS in aquaculture: applications and trends.

Science.gov (United States)

Rodrigues, Pedro M; Silva, Tomé S; Dias, Jorge; Jessen, Flemming

2012-07-19

Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million 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 of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological 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 nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteomics in uveal melanoma.

LENUS (Irish Health Repository)

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.

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Science.gov (United States)

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

Effects of Acanthopanax senticosus on Brain Injury Induced by Simulated Spatial Radiation in Mouse Model Based on Pharmacokinetics and Comparative Proteomics

Directory of Open Access Journals (Sweden)

Yingyu Zhou

2018-01-01

Full Text Available The active compounds in Acanthopanax senticosus (AS have different pharmacokinetic characteristics in mouse models. Cmax and AUC of Acanthopanax senticosus polysaccharides (ASPS were significantly reduced in radiation-injured mice, suggesting that the blood flow of mouse was blocked or slowed, due to the pathological state of ischemia and hypoxia, which are caused by radiation. In contrast, the ability of various metabolizing enzymes to inactivate, capacity of biofilm transport decrease, and lessening of renal blood flow accounts for radiation, resulting in the accumulation of syringin and eleutheroside E in the irradiated mouse. Therefore, there were higher pharmacokinetic parameters—AUC, MRT, and t1/2 of the two compounds in radiation-injured mouse, when compared with normal mouse. In order to investigate the intrinsic mechanism of AS on radiation injury, AS extract’s protective effects on brain, the main part of mouse that suffered from radiation, were explored. The function of AS extract in repressing expression changes of radiation response proteins in prefrontal cortex (PFC of mouse brain included tubulin protein family (α-, β-tubulin subunits, dihydropyrimidinase-related protein 2 (CRMP2, γ-actin, 14-3-3 protein family (14-3-3ζ, ε, heat shock protein 90β (HSP90β, and enolase 2. The results demonstrated the AS extract had positive effects on nerve cells’ structure, adhesion, locomotion, fission, and phagocytosis, through regulating various action pathways, such as Hippo, phagosome, PI3K/Akt (phosphatidylinositol 3 kinase/protein kinase B, Neurotrophin, Rap1 (Ras-related protein RAP-1A, gap junction glycolysis/gluconeogenesis, and HIF-1 (Hypoxia-inducible factor 1 signaling pathways to maintain normal mouse neurological activity. All of the results indicated that AS may be a promising alternative medicine for the treatment of radiation injury in mouse brain. It would be tested that whether the bioactive ingredients of AS could

Challenges for proteomics core facilities.

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Lilley, Kathryn S; Deery, Michael J; Gatto, Laurent

2011-03-01

Many analytical techniques have been executed by core facilities established within academic, pharmaceutical and other industrial institutions. The centralization of such facilities ensures a level of expertise and hardware which often cannot be supported by individual laboratories. The establishment of a core facility thus makes the technology available for multiple researchers in the same institution. Often, the services within the core facility are also opened out to researchers from other institutions, frequently with a fee being levied for the service provided. In the 1990s, with the onset of the age of genomics, there was an abundance of DNA analysis facilities, many of which have since disappeared from institutions and are now available through commercial sources. Ten years on, as proteomics was beginning to be utilized by many researchers, this technology found itself an ideal candidate for being placed within a core facility. We discuss what in our view are the daily challenges of proteomics core facilities. We also examine the potential unmet needs of the proteomics core facility that may also be applicable to proteomics laboratories which do not function as core facilities. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An individual urinary proteome analysis in normal human beings to define the minimal sample number to represent the normal urinary proteome

Directory of Open Access Journals (Sweden)

Liu Xuejiao

2012-11-01

Full Text Available Abstract Background The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics. Therefore, it is necessary to define the minimum number of individuals required for sampling to represent the normal urinary proteome. Methods In this study, inter-individual and inter-gender variations of urinary proteome were taken into consideration to achieve a representative database. An individual analysis was performed on overnight urine samples from 20 normal volunteers (10 males and 10 females by 1DLC/MS/MS. To obtain a representative result of each sample, a replicate 1DLCMS/MS analysis was performed. The minimal sample number was estimated by statistical analysis. Results For qualitative analysis, less than 5% of new proteins/peptides were identified in a male/female normal group by adding a new sample when the sample number exceeded nine. In addition, in a normal group, the percentage of newly identified proteins/peptides was less than 5% upon adding a new sample when the sample number reached 10. Furthermore, a statistical analysis indicated that urinary proteomes from normal males and females showed different patterns. For quantitative analysis, the variation of protein abundance was defined by spectrum count and western blotting methods. And then the minimal sample number for quantitative proteomic analysis was identified. Conclusions For qualitative analysis, when considering the inter-individual and inter-gender variations, the minimum sample number is 10 and requires a balanced number of males and females in order to obtain a representative normal human urinary proteome. For quantitative analysis, the minimal sample number is much greater than that for qualitative analysis and depends on the experimental methods used for quantification.

Proteomic approaches in research of cyanobacterial photosynthesis.

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Battchikova, Natalia; Angeleri, Martina; Aro, Eva-Mari

2015-10-01

Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

Science.gov (United States)

Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Automation, parallelism, and robotics for proteomics.

Science.gov (United States)

Alterovitz, Gil; Liu, Jonathan; Chow, Jijun; Ramoni, Marco F

2006-07-01

The speed of the human genome project (Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C. et al., Nature 2001, 409, 860-921) was made possible, in part, by developments in automation of sequencing technologies. Before these technologies, sequencing was a laborious, expensive, and personnel-intensive task. Similarly, automation and robotics are changing the field of proteomics today. Proteomics is defined as the effort to understand and characterize proteins in the categories of structure, function and interaction (Englbrecht, C. C., Facius, A., Comb. Chem. High Throughput Screen. 2005, 8, 705-715). As such, this field nicely lends itself to automation technologies since these methods often require large economies of scale in order to achieve cost and time-saving benefits. This article describes some of the technologies and methods being applied in proteomics in order to facilitate automation within the field as well as in linking proteomics-based information with other related research areas.

Expanding the bovine milk proteome through extensive fractionation

DEFF Research Database (Denmark)

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne

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...... 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...... 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...

Spermatogenesis in mammals: proteomic insights.

Science.gov (United States)

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

Polyphemus, Odysseus and the ovine milk proteome.

Science.gov (United States)

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.

Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS

Science.gov (United States)

Zeng, Xuemei; Hood, Brian L.; Zhao, Ting; Conrads, Thomas P.; Sun, Mai; Gopalakrishnan, Vanathi; Grover, Himanshu; Day, Roger S.; Weissfeld, Joel L.; Wilson, David O.; Siegfried, Jill M.; Bigbee, William L.

2011-01-01

Introduction Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit. Methods We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays. Results A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (pbiomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection. PMID:21304412

The Coming Age of Complete, Accurate, and Ubiquitous Proteomes

DEFF Research Database (Denmark)

Mann, M.; Kulak, N.A.; Nagaraj, N.

2013-01-01

High-resolution mass spectrometry (MS)-based proteomics has progressed tremendously over the years. For model organisms like yeast, we can now quantify complete proteomes in just a few hours. Developments discussed in this Perspective will soon enable complete proteome analysis of mammalian cells...

Glycoproteins and Glycosylation Site Assignments in Cereal seed Proteomes

DEFF Research Database (Denmark)

Dedvisitsakul, Plaipol

The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosy......The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications...

Mathematical biodescriptors of proteomics maps: background and applications.

Science.gov (United States)

Basak, Subhash C; Gute, Brian D

2008-05-01

This article reviews recent developments in the formulation and application of biodescriptors to characterize proteomics maps. Such biodescriptors can be derived by applying techniques from discrete mathematics (graph theory, linear algebra and information theory). This review focuses on the development of biodescriptors for proteomics maps derived from 2D gel electrophoresis. Preliminary results demonstrated that such descriptors have a reasonable ability to differentiate between proteomics patterns that result from exposure to closely related individual chemicals and complex mixtures, such as the jet fuel JP-8. Further research is required to evaluate the utility of these proteomics-based biodescriptors for drug discovery and predictive toxicology.

Characterization of the porcine synovial fluid proteome and a comparison to the plasma proteome

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Barnaby, Omar; Steen, Hanno

2015-01-01

Synovial fluid is present in all joint cavities, and protects the articular cartilage surfaces in large by lubricating the joint, thus reducing friction. Several studies have described changes in the protein composition of synovial fluid in patients with joint disease. However, the protein concen...... 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....

Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

Science.gov (United States)

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

Proteomics: Protein Identification Using Online Databases

Science.gov (United States)

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…

Modification-specific proteomics in plant biology

DEFF Research Database (Denmark)

Ytterberg, A Jimmy; Jensen, Ole N

2010-01-01

and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM...

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia

OpenAIRE

Ihnatko, Robert; Post, Claes; Blomqvist, Anders

2013-01-01

Background: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain’s metabolic control centre. Methods: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed litterma...

Ascorbic acid effects on in vitro maturation of mouse oocyte with or ...

African Journals Online (AJOL)

Ascorbic acid has long been associated with fertility. This study was designed to determine the effects of ascorbic acid on in vitro maturation of mouse oocyte with or without cumulus cells. In this study, 508 denuded oocytes (DOs) and 527 cumulus–oocyte complexes (COCs) from mice stimulated with pregnant mare's serum ...

Modifying effect of COMT gene polymorphism and a predictive role for proteomics analysis in children's intelligence in endemic fluorosis area in Tianjin, China.

Science.gov (United States)

Zhang, Shun; Zhang, Xiaofei; Liu, Hongliang; Qu, Weidong; Guan, Zhizhong; Zeng, Qiang; Jiang, Chunyang; Gao, Hui; Zhang, Cheng; Lei, Rongrong; Xia, Tao; Wang, Zhenglun; Yang, Lu; Chen, Yihu; Wu, Xue; Cui, Yushan; Yu, Linyu; Wang, Aiguo

2015-04-01

Cumulative fluoride exposure has adverse influences on children's intelligence quotient (IQ). In addition, catechol-O-methyltransferase (COMT) gene Val158Met polymorphism (rs4680) is associated with cognitive performance. This study aimed to evaluate the associations of COMT polymorphism and alterations of protein profiles with children's intelligence in endemic fluorosis area. We recruited 180 schoolchildren (10-12 years old) from high fluoride exposure (1.40 mg/l) and control areas (0.63 mg/l) in Tianjin City, China. The children's IQ, fluoride contents in drinking water (W-F), serum (S-F), and urine (U-F); serum thyroid hormone levels, COMT Val158Met polymorphism, and plasma proteomic profiling were determined. Significant high levels of W-F, S-F, U-F, along with poor IQ scores were observed in the high fluoride exposure group compared with those in control (all P intelligence, whereas the COMT polymorphism may increase the susceptibility to the deficits in IQ due to fluoride exposure. Moreover, the proteomic analysis can provide certain basis for identifying the early biological markers of fluorosis among children. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Biomarker discovery in mass spectrometry-based urinary proteomics.

Science.gov (United States)

Thomas, Samuel; Hao, Ling; Ricke, William A; Li, Lingjun

2016-04-01

Urinary proteomics has become one of the most attractive topics in disease biomarker discovery. MS-based proteomic analysis has advanced continuously and emerged as a prominent tool in the field of clinical bioanalysis. However, only few protein biomarkers have made their way to validation and clinical practice. Biomarker discovery is challenged by many clinical and analytical factors including, but not limited to, the complexity of urine and the wide dynamic range of endogenous proteins in the sample. This article highlights promising technologies and strategies in the MS-based biomarker discovery process, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics. Different proteomics approaches are discussed, and progresses in maximizing urinary proteome coverage and standardization are emphasized in this review. MS-based urinary proteomics has great potential in the development of noninvasive diagnostic assays in the future, which will require collaborative efforts between analytical scientists, systems biologists, and clinicians. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome

Science.gov (United States)

Kosová, Klára; Vítámvás, Pavel; Urban, Milan O.; Prášil, Ilja T.; Renaut, Jenny

2018-01-01

HIGHLIGHTS: Major environmental and genetic factors determining stress-related protein abundance are discussed.Major aspects of protein biological function including protein isoforms and PTMs, cellular localization and protein interactions are discussed.Functional diversity of protein isoforms and PTMs is discussed. Abiotic stresses reveal profound impacts on plant proteomes including alterations in protein relative abundance, cellular localization, post-transcriptional and post-translational modifications (PTMs), protein interactions with other protein partners, and, finally, protein biological functions. The main aim of the present review is to discuss the major factors determining stress-related protein accumulation and their final biological functions. A dynamics of stress response including stress acclimation to altered ambient conditions and recovery after the stress treatment is discussed. The results of proteomic studies aimed at a comparison of stress response in plant genotypes differing in stress adaptability reveal constitutively enhanced levels of several stress-related proteins (protective proteins, chaperones, ROS scavenging- and detoxification-related enzymes) in the tolerant genotypes with respect to the susceptible ones. Tolerant genotypes can efficiently adjust energy metabolism to enhanced needs during stress acclimation. Stress tolerance vs. stress susceptibility are relative terms which can reflect different stress-coping strategies depending on the given stress treatment. The role of differential protein isoforms and PTMs with respect to their biological functions in different physiological constraints (cellular compartments and interacting partners) is discussed. The importance of protein functional studies following high-throughput proteome analyses is presented in a broader context of plant biology. In summary, the manuscript tries to provide an overview of the major factors which have to be considered when interpreting data from proteomic

Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor

Science.gov (United States)

Wu, Yingjie; Sun, Hui; Basta-Pljakic, Jelena; Cardoso, Luis; Kennedy, Oran D; Jasper, Hector; Domené, Horacio; Karabatas, Liliana; Guida, Clara; Schaffler, Mitchell B; Rosen, Clifford J; Yakar, Shoshana

2013-01-01

States of growth hormone (GH) resistance, such those observed in Laron’s dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKOHIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1 allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron’s syndrome does not achieve full skeletal growth. PMID:23456957

Proteomic approach to nanotoxicity.

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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.

The quest of the human proteome and the missing proteins: digging deeper.

Science.gov (United States)

Reddy, Panga Jaipal; Ray, Sandipan; Srivastava, Sanjeeva

2015-05-01

Given the diverse range of transcriptional and post-transcriptional mechanisms of gene regulation, the estimates of the human proteome is likely subject to scientific surprises as the field of proteomics has gained momentum worldwide. In this regard, the establishment of the "Human Proteome Draft" using high-resolution mass spectrometry (MS), tissue microarrays, and immunohistochemistry by three independent research groups (laboratories of Pandey, Kuster, and Uhlen) accelerated the pace of proteomics research. The Chromosome Centric Human Proteome Project (C-HPP) has taken initiative towards the completion of the Human Proteome Project (HPP) so as to understand the proteomics correlates of common complex human diseases and biological diversity, not to mention person-to-person and population differences in response to drugs, nutrition, vaccines, and other health interventions and host-environment interactions. Although high-resolution MS-based and antibody microarray approaches have shown enormous promises, we are still unable to map the whole human proteome due to the presence of numerous "missing proteins." In December 2014, at the Indian Institute of Technology Bombay, Mumbai the 6(th) Annual Meeting of the Proteomics Society, India (PSI) and the International Proteomics Conference was held. As part of this interdisciplinary summit, a panel discussion session on "The Quest of the Human Proteome and Missing Proteins" was organized. Eminent scientists in the field of proteomics and systems biology, including Akhilesh Pandey, Gilbert S. Omenn, Mark S. Baker, and Robert L. Mortiz, shed light on different aspects of the human proteome drafts and missing proteins. Importantly, the possible reasons for the "missing proteins" in shotgun MS workflow were identified and debated by experts as low tissue expression, lack of enzymatic digestion site, or protein lost during extraction, among other contributing factors. To capture the missing proteins, the experts' collective

Serum erythropoietin levels by radioimmunoassay in polycythaemia

Energy Technology Data Exchange (ETDEWEB)

Birgegaard, G.; Miller, O.; Caro, J.; Erslev, A. (Cardeza Foundation for Hematological Research, Jefferson University, Philadelphia, Pa.)

1982-01-01

A radioimmunoassay (RIA) method for erythropoietin (Epo) was developed and validated against the polycythaemic mouse assay. The correlation was good, with a r=0.94. Several other criteria of specificity were also filled by the RIA, which had a lower detection limit of 5 mU/ml. The mean serum-Epo level in 6 patients with secondary polycythaemia, 50.2 +- 26.2 mU/ml, was significantly higher than in a group of 11 normal subjects, 28.7 +- 7.2 mU/ml (P<0.0002). However, the Epo level in 31 polycythaemia vera (PV) patients, M = 21.9 +- 6.6 mU/ml, was not significantly different from normal (P = 0.006). Since previous studies with bioassay of heat-treated and concentrated plasma samples have shown a decreased serum-Epo level in PV, Epo levels were measured before and after heat treatment and concentration of samples from normals and polycythaemics. It was found that the levels of immunoreactive material increased after heat treatment and 40 times concentration in samples from normals and patients with secondary polycythaemias, but decreased in PV. We conclude that the Epo levels in serum in the low range measured by our and previous RIA:s probably are not true Epo levels but are partly due to an unspecific serum effect, that was removed by heat treatment.

Clinical proteomics: Current status, challenges, and future perspectives

Directory of Open Access Journals (Sweden)

Shyh-Horng Chiou

2011-01-01

Full Text Available This account will give an overview and evaluation of the current advances in mass spectrometry (MS-based proteomics platforms and technology. A general review of some background information concerning the application of these methods in the characterization of molecular sizes and related protein expression profiles associated with different types of cells under varied experimental conditions will be presented. It is intended to provide a concise and succinct overview to those clinical researchers first exposed to this foremost powerful methodology in modern life sciences of postgenomic era. Proteomic characterization using highly sophisticated and expensive instrumentation of MS has been used to characterize biological samples of complex protein mixtures with vastly different protein structure and composition. These systems are then used to highlight the versatility and potential of the MS-based proteomic strategies for facilitating protein expression analysis of various disease-related organisms or tissues of interest. Major MS-based strategies reviewed herein include (1 matrix-assisted laser desorption ionization-MS and electron-spray ionization proteomics; (2 one-dimensional or two-dimensional gel-based proteomics; (3 gel-free shotgun proteomics in conjunction with liquid chromatography/tandem MS; (4 Multiple reaction monitoring coupled tandem MS quantitative proteomics and; (5 Phosphoproteomics based on immobilized metal affinity chromatography and liquid chromatography-MS/MS.