Transcriptome and proteome data reveal candidate genes for pollinator attraction in sexually deceptive orchids.

Science.gov (United States)

Sedeek, Khalid E M; Qi, Weihong; Schauer, Monica A; Gupta, Alok K; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P; Schlüter, Philipp M

2013-01-01

Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of

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.

Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics

DEFF Research Database (Denmark)

Yang, Fen; Braga, Marcella Nunes de Melo; Larsen, Martin Røssel

2013-01-01

The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat-S. tritici interaction, we performed a time-course study of S. tritici infection...... in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during...... compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger...

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

DEFF Research Database (Denmark)

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

2015-01-01

and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions...

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

Science.gov (United States)

Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

2014-12-01

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptome and Proteome Studies Reveal Candidate Attachment Genes during the Development of the Barnacle Amphibalanus Amphitrite

KAUST Repository

Al-Aqeel, Sarah; Ryu, Tae Woo; Zhang, Huoming; Chandramouli, Kondethimmanahalli; Ravasi, Timothy

2016-01-01

The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41%. We identified 65,784 expressed contigs, and a total of 1387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development. Proteomics data are available via ProteomeXchange with identifier PXD004679.

Transcriptome and Proteome Studies Reveal Candidate Attachment Genes during the Development of the Barnacle Amphibalanus Amphitrite

KAUST Repository

Al-Aqeel, Sarah

2016-09-21

The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41%. We identified 65,784 expressed contigs, and a total of 1387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development. Proteomics data are available via ProteomeXchange with identifier PXD004679.

Proteomic properties reveal phyloecological clusters of Archaea.

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Nela Nikolic

Full Text Available In this study, we propose a novel way to describe the variety of environmental adaptations of Archaea. We have clustered 57 Archaea by using a non-redundant set of proteomic features, and verified that the clusters correspond to environmental adaptations to the archaeal habitats. The first cluster consists dominantly of hyperthermophiles and hyperthermoacidophilic aerobes. The second cluster joins together halophilic and extremely halophilic Archaea, while the third cluster contains mesophilic (mostly methanogenic Archaea together with thermoacidophiles. The non-redundant subset of proteomic features was found to consist of five features: the ratio of charged residues to uncharged, average protein size, normalized frequency of beta-sheet, normalized frequency of extended structure and number of hydrogen bond donors. We propose this clustering to be termed phyloecological clustering. This approach could give additional insights into relationships among archaeal species that may be hidden by sole phylogenetic analysis.

Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas.

Science.gov (United States)

Timmins-Schiffman, Emma; Coffey, William D; Hua, Wilber; Nunn, Brook L; Dickinson, Gary H; Roberts, Steven B

2014-11-03

Ocean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end-of-century open ocean pH reductions. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different p CO2 levels for four weeks: 400 μatm (pH 8.0), 800 μatm (pH 7.7), 1000 μatm (pH 7.6), or 2800 μatm (pH 7.3). At the end of the four week exposure period, oysters in all four p CO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated p CO2. Elevated p CO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with p CO2, with numerous processes significantly affected by mechanical stimulation at high versus low p CO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835). Oyster physiology is significantly altered by exposure to elevated p CO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of p CO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.

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

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Nielsen Niels

2010-02-01

Full Text Available Abstract Background 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 of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. Results The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the

Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

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Nicos Angelopoulos

2016-06-01

Full Text Available Tyrosine kinases (TKs play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015 [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015 [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

Science.gov (United States)

Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

2012-01-10

The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal

OpenAIRE

Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

2015-01-01

A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transf...

Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

KAUST Repository

Mu, Huawei; Sun, Jin; Cheung, Siu Gin; Fang, Ling; Zhou, Haiyun; Luan, Tiangang; Zhang, Huoming; Wong, Chris K.C.; Qiu, Jian-Wen

2017-01-01

Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic

Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

KAUST Repository

Mu, Huawei

2017-11-06

Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic

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

Science.gov (United States)

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.

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger.

Science.gov (United States)

Manzanares-Miralles, Lara; Sarikaya-Bayram, Özlem; Smith, Elizabeth B; Dolan, Stephen K; Bayram, Özgür; Jones, Gary W; Doyle, Sean

2016-01-10

Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus, which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p=0.0018) required for homocysteine generation from S-adenosylhomocysteine (SAH), and spermidine synthase (p=0.0068), involved in the recycling of Met, was observed. Analysis of Met-related metabolites revealed significant increases in the levels of Met and adenosine, in correlation with proteomic data. Methyltransferase MT-II is responsible for bisthiobis(methylthio)gliotoxin (BmGT) formation, deletion of MT-II abolished BmGT formation and led to increased GT sensitivity in A. niger. Proteomic analysis also revealed that GT exposure also significantly (pniger. Thus, it provides new opportunities to exploit the response of GT-naïve fungi to GT. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

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Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

Directory of Open Access Journals (Sweden)

Kosalková Katarina

2012-01-01

Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

Science.gov (United States)

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

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

Science.gov (United States)

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

Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies.

Science.gov (United States)

Al Shweiki, Mhd Rami; Oeckl, Patrick; Steinacker, Petra; Hengerer, Bastian; Schönfeldt-Lecuona, Carlos; Otto, Markus

2017-06-01

Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates. Areas covered: The review includes the human studies found by a PubMed search using the following terms: 'depression cerebrospinal fluid biomarker', 'major depression biomarker CSF', 'depression CSF biomarker', 'proteomics depression', 'proteomics biomarkers in depression', 'proteomics CSF biomarker in depression', and 'major depressive disorder CSF'. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies. Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.

Proteomic Characterization of Armillaria mellea Reveals Oxidative Stress Response Mechanisms and Altered Secondary Metabolism Profiles

Directory of Open Access Journals (Sweden)

Cassandra Collins

2017-09-01

Full Text Available Armillaria mellea is a major plant pathogen. Yet, the strategies the organism uses to infect susceptible species, degrade lignocellulose and other plant material and protect itself against plant defences and its own glycodegradative arsenal are largely unknown. Here, we use a combination of gel and MS-based proteomics to profile A. mellea under conditions of oxidative stress and changes in growth matrix. 2-DE and LC-MS/MS were used to investigate the response of A. mellea to H2O2 and menadione/FeCl3 exposure, respectively. Several proteins were detected with altered abundance in response to H2O2, but not menadione/FeCl3 (i.e., valosin-containing protein, indicating distinct responses to these different forms of oxidative stress. One protein, cobalamin-independent methionine synthase, demonstrated a common response in both conditions, which may be a marker for a more general stress response mechanism. Further changes to the A. mellea proteome were investigated using MS-based proteomics, which identified changes to putative secondary metabolism (SM enzymes upon growth in agar compared to liquid cultures. Metabolomic analyses revealed distinct profiles, highlighting the effect of growth matrix on SM production. This establishes robust methods by which to utilize comparative proteomics to characterize this important phytopathogen.

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.

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.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

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Zai, Xiaodong; Yang, Qiaoling; Yin, Ying; Li, Ruihua; Qian, Mengying; Zhao, Taoran; Li, Yaohui; Zhang, Jun; Fu, Ling; Xu, Junjie; Chen, Wei

2017-01-01

Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic

Fish gut-liver immunity during homeostasis or inflammation revealed by integrative transcriptome and proteome studies

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Wu, Nan; Song, Yu-Long; Wang, Bei; Zhang, Xiang-Yang; Zhang, Xu-Jie; Wang, Ya-Li; Cheng, Ying-Yin; Chen, Dan-Dan; Xia, Xiao-Qin; Lu, Yi-Shan; Zhang, Yong-An

2016-11-01

The gut-associated lymphoid tissue, connected with liver via bile and blood, constructs a local immune environment of both defense and tolerance. The gut-liver immunity has been well-studied in mammals, yet in fish remains largely unknown, even though enteritis as well as liver and gallbladder syndrome emerged as a limitation in aquaculture. In this study, we performed integrative bioinformatic analysis for both transcriptomic (gut and liver) and proteomic (intestinal mucus and bile) data, in both healthy and infected tilapias. We found more categories of immune transcripts in gut than liver, as well as more adaptive immune in gut meanwhile more innate in liver. Interestingly reduced differential immune transcripts between gut and liver upon inflammation were also revealed. In addition, more immune proteins in bile than intestinal mucus were identified. And bile probably providing immune effectors to intestinal mucus upon inflammation was deduced. Specifically, many key immune transcripts in gut or liver as well as key immune proteins in mucus or bile were demonstrated. Accordingly, we proposed a hypothesized profile of fish gut-liver immunity, during either homeostasis or inflammation. Current data suggested that fish gut and liver may collaborate immunologically while keep homeostasis using own strategies, including potential unique mechanisms.

Proteomic investigation of aphid honeydew reveals an unexpected diversity of proteins.

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Ahmed Sabri

Full Text Available Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora. Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu, and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective.

Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typimurium in response to infection-like conditions

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Ansong, Charles; Wu, Si; Meng, Da; Liu, Xiaowen; Brewer, Heather M.; Kaiser, Brooke LD; Nakayasu, Ernesto S.; Cort, John R.; Pevzner, Pavel A.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; Pasa-Tolic, Ljiljana

2013-06-18

Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Bottom-up proteomic approaches often lead to loss of critical information about an endogenous protein’s actual state due to post translational modifications (PTMs) and other processes. Top-down approaches that involve analysis of the intact protein can address this concern but present significant analytical challenges related to the separation quality needed, measurement sensitivity, and speed that result in low throughput and limited coverage. Here we used single-dimension ultra high pressure liquid chromatography mass spectrometry to investigate the comprehensive ‘intact’ proteome of the Gram negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1665 proteoforms generated by PTMs, representing the largest microbial top-down dataset reported to date. Our analysis not only confirmed several previously recognized aspects of Salmonella biology and bacterial PTMs in general, but also revealed several novel biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions, which was corroborated by changes in corresponding biosynthetic pathways. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents to our knowledge the first report of S-cysteinylation in Gram negative bacteria. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.

Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

International Nuclear Information System (INIS)

Jami, Mohammad-Saeid; Huang, Xin; Peng, Hong; Fu, Kai; Li, Yan; Singh, Rakesh K; Ding, Shi-Jian; Hou, Jinxuan; Liu, Miao; Varney, Michelle L; Hassan, Hesham; Dong, Jixin; Geng, Liying; Wang, Jing; Yu, Fang

2014-01-01

KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it

Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

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Jaiswal Dinesh Kumar

2012-10-01

Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

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Xiaodong Zai

2017-11-01

Full Text Available Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

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Eric K Johnson

Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

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Qingzhu Hua

2016-09-01

Full Text Available Red dragon fruit or red pitaya (Hylocereus polyrhizus is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

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Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

2005-07-01

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility

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Ashok Agarwal

2016-01-01

Full Text Available To study the major differences in the distribution of spermatozoa proteins in infertile men with varicocele by comparative proteomics and validation of their level of expression. The study-specific estimates for each varicocele outcome were combined to identify the proteins involved in varicocele-associated infertility in men irrespective of stage and laterality of their clinical varicocele. Expression levels of 5 key proteins (PKAR1A, AK7, CCT6B, HSPA2, and ODF2 involved in stress response and sperm function including molecular chaperones were validated by Western blotting. Ninety-nine proteins were differentially expressed in the varicocele group. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in the varicocele group. Key protein functions affected in the varicocele group were spermatogenesis, sperm motility, and mitochondrial dysfunction, which were further validated by Western blotting, corroborating the proteomics analysis. Varicocele is essentially a state of energy deprivation, hypoxia, and hyperthermia due to impaired blood supply, which is corroborated by down-regulation of lipid metabolism, mitochondrial electron transport chain, and Krebs cycle enzymes. To corroborate the proteomic analysis, expression of the 5 identified proteins of interest was validated by Western blotting. This study contributes toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters.

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

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Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

2016-06-30

Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells

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Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

2016-01-01

to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...

Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta "Gold Standard" Leaves.

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Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

2016-03-08

Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta "Gold Standard" is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta "Gold Standard", as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta "Gold Standard". For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.

2D-DIGE analysis of mango (Mangifera indica L.) fruit reveals major proteomic changes associated with ripening.

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Andrade, Jonathan de Magalhães; Toledo, Tatiana Torres; Nogueira, Silvia Beserra; Cordenunsi, Beatriz Rosana; Lajolo, Franco Maria; do Nascimento, João Roberto Oliveira

2012-06-18

A comparative proteomic investigation between the pre-climacteric and climacteric mango fruits (cv. Keitt) was performed to identify protein species with variable abundance during ripening. Proteins were phenol-extracted from fruits, cyanine-dye-labeled, and separated on 2D gels at pH 4-7. Total spot count of about 373 proteins spots was detected in each gel and forty-seven were consistently different between pre-climacteric and climacteric fruits and were subjected to LC-MS/MS analysis. Functional classification revealed that protein species involved in carbon fixation and hormone biosynthesis decreased during ripening, whereas those related to catabolism and the stress-response, including oxidative stress and abiotic and pathogen defense factors, accumulated. In relation to fruit quality, protein species putatively involved in color development and pulp softening were also identified. This study on mango proteomics provides an overview of the biological processes that occur during ripening. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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Helle H Nielsen

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

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.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

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Monique Ramos de Oliveira Trugilho

2017-05-01

Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome

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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 proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

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Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several

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

DEFF Research Database (Denmark)

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 ...

Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

DEFF Research Database (Denmark)

Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

2006-01-01

Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics.

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Yang, Fen; Melo-Braga, Marcella N; Larsen, Martin R; Jørgensen, Hans J L; Palmisano, Giuseppe

2013-09-01

The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat-S. tritici interaction, we performed a time-course study of S. tritici infection in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger accumulation of signal molecules, including calcium, H2O2, NO, and sugars, in the resistant than in the susceptible cultivar in response to the infection. Additionally, 31 proteins and 5 phosphoproteins from the pathogen were identified, including metabolic proteins and signaling proteins such as GTP-binding proteins, 14-3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a comprehensive

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

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Candida Vannini

Full Text Available Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

In vivo Host-Pathogen Interaction as Revealed by Global Proteomic Profiling of Zebrafish Larvae

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Francisco Díaz-Pascual

2017-07-01

Full Text Available The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1 and the host (zebrafish. We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, were exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, and cellular responses to antibiotic and starvation were enriched exclusively after exposure by

Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

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Swelm, Rachel P.L. van [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Laarakkers, Coby M.M. [Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Pertijs, Jeanne C.L.M.; Verweij, Vivienne; Masereeuw, Rosalinde [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Russel, Frans G.M., E-mail: F.Russel@pharmtox.umcn.nl [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands)

2013-06-01

Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75 mg/kg bw DF by oral gavage and 24 h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p < 0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p < 0.001 and p < 0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p < 0.001) and protein expression of PCNA (p < 0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24 h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury. - Highlights: • The urinary proteome shows biological processes involved in adverse drug reactions. • Urine proteins of DF-treated mice relate to kidney injury rather than liver injury. • Liver regeneration, not liver injury, is apparent 24h after oral DF administration. • Pretreatment with LPS does not enhance DF-induced liver injury in mice.

iTRAQ-based proteomic analysis reveals alterations in the liver induced by restricted meal frequency in a pig model.

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Liu, Jingbo; Liu, Zhengqun; Chen, Liang; Zhang, Hongfu

2016-01-01

The present study was conducted to investigate the effects of meal frequency on metabolite levels in pig plasma and hepatic proteome by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Twenty-four pigs (60.7 ± 1.0 kg) consumed the same amount of feed either in 2 (M2, n = 12) or 12 (M12, n = 12) meals per day. After an 8-wk feeding period, plasma concentrations of metabolites and hormones, hepatic biochemical traits, and proteome (n = 4 per group) were measured. Pigs on the M12 regimen had lower average daily gain and gain-to-feed ratio than pigs fed the M2 regimen. The M2 regimen resulted in lower total lipid, glycogen, and triacylglycerol content in the liver and circulating triacylglycerol concentration than that in the M12 pigs. The metabolic hormone concentrations were not affected by meal frequency, with the exception of elevated fibroblast growth factor 21 concentrations in the M2 regimen compared with the M12 regimen. The iTRAQ-based proteomic analysis revealed 35 differentially expressed proteins in the liver between pigs fed two and 12 meals per day, and these differentially expressed proteins were involved in the regulation of general biological process such as glucose and energy metabolism, lipid metabolism, protein and amino acid metabolism, stress response, and cell redox homeostasis. Altogether, the proteomic results provide insights into the mechanism mediating the beneficial effects of restricted meal frequency on the metabolic fitness. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

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Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

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Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

2017-10-06

Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

2014-01-01

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang

2014-06-11

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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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

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer*

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Chen, Chien-Lun; Chung, Ting; Wu, Chih-Ching; Ng, Kwai-Fong; Yu, Jau-Song; Tsai, Cheng-Han; Chang, Yu-Sun; Liang, Ying; Tsui, Ke-Hung; Chen, Yi-Ting

2015-01-01

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins—SLC3A2, STMN1, and TAGLN2—in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant

Transcriptome and proteome studies reveal candidate attachment genes during the development of the barnacle Amphibalanus Amphitrite

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Sarah Al-Aqeel

2016-09-01

Full Text Available The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI and cyprid from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41‰. We identified 65,784 expressed contigs, and a total of 1,387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development.

Proteomic Technologies for the Study of Osteosarcoma

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Stephanie D. Byrum

2012-01-01

Full Text Available Osteosarcoma is the most common primary bone cancer of children and is established during stages of rapid bone growth. The disease is a consequence of immature osteoblast differentiation, which gives way to a rapidly synthesized incompletely mineralized and disorganized bone matrix. The mechanism of osteosarcoma tumorogenesis is poorly understood, and few proteomic studies have been used to interrogate the disease thus far. Accordingly, these studies have identified proteins that have been known to be associated with other malignancies, rather than being osteosarcoma specific. In this paper, we focus on the growing list of available state-of-the-art proteomic technologies and their specific application to the discovery of novel osteosarcoma diagnostic and therapeutic targets. The current signaling markers/pathways associated with primary and metastatic osteosarcoma that have been identified by early-stage proteomic technologies thus far are also described.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

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Wan, Huafang; Cui, Yixin; Ding, Yijuan; Mei, Jiaqin; Dong, Hongli; Zhang, Wenxin; Wu, Shiqi; Liang, Ying; Zhang, Chunyu; Li, Jiana; Xiong, Qing; Qian, Wei

2016-01-01

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola ( Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

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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

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

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Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

2016-02-05

Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

Personalized Proteome Profiles of Healthy and Tumor Human Colon Organoids Reveal Both Individual Diversity and Basic Features of Colorectal Cancer.

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Cristobal, Alba; van den Toorn, Henk W P; van de Wetering, Marc; Clevers, Hans; Heck, Albert J R; Mohammed, Shabaz

2017-01-03

Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

DEFF Research Database (Denmark)

Francavilla, Chiara; Papetti, Moreno; Rigbolt, Kristoffer T G

2016-01-01

, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We...... identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF......-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling....

Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon.

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Sousa, Leijiane F; Portes-Junior, José A; Nicolau, Carolina A; Bernardoni, Juliana L; Nishiyama, Milton Y; Amazonas, Diana R; Freitas-de-Sousa, Luciana A; Mourão, Rosa Hv; Chalkidis, Hipócrates M; Valente, Richard H; Moura-da-Silva, Ana M

2017-04-21

Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA 2 s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA 2 s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not

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

Science.gov (United States)

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

2012-11-02

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

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

Energy Technology Data Exchange (ETDEWEB)

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.

Targeted proteomics guided by label-free global proteome analysis in saliva reveal transition signatures from health to periodontal disease.

Science.gov (United States)

Bostanci, Nagihan; Selevsek, Nathalie; Wolski, Witold; Grossmann, Jonas; Bao, Kai; Wahlander, Asa; Trachsel, Christian; Schlapbach, Ralph; Özturk, Veli Özgen; Afacan, Beral; Emingil, Gulnur; Belibasakis, Georgios N

2018-04-02

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. We carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n=67, health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n=82). The LFQ platform led to the discovery of 119 proteins with at least two-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1), with maximum area under the receiver operating curve >0.97. This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum

Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

Directory of Open Access Journals (Sweden)

Jackson Champer

2016-01-01

Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-01-01

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

Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium

Energy Technology Data Exchange (ETDEWEB)

Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar, E-mail: aptesk@barc.gov.in

2017-01-15

Highlights: • Response of two native cyanobacterial strains to uranium exposure was studied. • Anabaena L-31 exhibited higher tolerance to uranium as compared to Anabaena 7120. • Uranium exposure differentially affected the proteome profiles of the two strains. • Anabaena L-31 showed better sustenance of photosynthesis and carbon metabolism. • Anabaena L-31 displayed superior oxidative stress defense than Anabaena 7120. - Abstract: Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD{sub 50} dose), following 3 h exposure to 75 μM and 200 μM uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120. Significance: Uranium responsive proteome modulations in two nitrogen-fixing strains of Anabaena, native to Indian paddy fields, revealed that rapid adaptation to better oxidative stress management, and maintenance of metabolic and energy homeostasis underlies superior uranium tolerance of Anabaena sp. strain L-31 compared to Anabaena sp. strain PCC 7120.

Proteomic studies of drought stress response in Fabaceae

Directory of Open Access Journals (Sweden)

Tanja ZADRAŽNIK

2015-11-01

Full Text Available Drought stress is a serious threat to crop production that influences plant growth and development and subsequently causes reduced quantity and quality of the yield. Plant stress induces changes in cell metabolism, which includes differential expression of proteins. Proteomics offer a powerful approach to analyse proteins involved in drought stress response of plants. Analyses of changes in protein abundance of legumes under drought stress are very important, as legumes play an important role in human and animal diet and are often exposed to drought. The presented results of proteomic studies of selected legumes enable better understanding of molecular mechanisms of drought stress response. The study of drought stress response of plants with proteomic approach may contribute to the development of potential drought-response markers and to the development of drought-tolerant cultivars of different legume crop species.

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

Directory of Open Access Journals (Sweden)

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.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.

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Whittington, Emma; Forsythe, Desiree; Borziak, Kirill; Karr, Timothy L; Walters, James R; Dorus, Steve

2017-12-02

Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Our results identify a burst of genetic novelty

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

OpenAIRE

Manzanares-Miralles, Lara; Bayram, Ozgur; Sarikaya-Bayram, Ozlem; Smith, Elizabeth B.; Dolan, Stephen K.; Jones, Gary W.; Doyle, Sean

2016-01-01

Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus,which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p = 0.0018) ...

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

KAUST Repository

Zhang, Huoming

2010-07-02

Human monocytes\\' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

KAUST Repository

Zhang, Huoming; Zhao, Changqing; Li, Xin; Zhu, Yi; Gan, Chee Sian; Wang, Yong; Ravasi, Timothy; Qian, Pei-Yuan; Wong, Siew Cheng; Sze, Siu Kwan

2010-01-01

Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

Long-term heat stress induces the inflammatory response in dairy cows revealed by plasma proteome analysis.

Science.gov (United States)

Min, Li; Zheng, Nan; Zhao, Shengguo; Cheng, Jianbo; Yang, Yongxin; Zhang, Yangdong; Yang, Hongjian; Wang, Jiaqi

2016-03-04

In this work we employed a comparative proteomic approach to evaluate seasonal heat stress and investigate proteomic alterations in plasma of dairy cows. Twelve lactating Holstein dairy cows were used and the treatments were: heat stress (n = 6) in hot summer (at the beginning of the moderate heat stress) and no heat stress (n = 6) in spring natural ambient environment, respectively. Subsequently, heat stress treatment lasted 23 days (at the end of the moderate heat stress) to investigate the alterations of plasma proteins, which might be employed as long-term moderate heat stress response in dairy cows. Changes in plasma proteins were analyzed by two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Analysis of the properties of the identified proteins revealed that the alterations of plasma proteins were related to inflammation in long-term moderate heat stress. Furthermore, the increase in plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) directly demonstrated that long-term moderate heat stress caused an inflammatory response in dairy cows. Copyright © 2016 Elsevier Inc. All rights reserved.

Water deficit mechanisms in perennial shrubs Cerasus humilis leaves revealed by physiological and proteomic analyses.

Science.gov (United States)

Yin, Zepeng; Ren, Jing; Zhou, Lijuan; Sun, Lina; Wang, Jiewan; Liu, Yulong; Song, Xingshun

2016-01-01

Drought (Water deficit, WD) poses a serious threat to extensively economic losses of trees throughout the world. Chinese dwarf cherry ( Cerasus humilis ) is a good perennial plant for studying the physiological and sophisticated molecular network under WD. The aim of this study is to identify the effect of WD on C. humilis through physiological and global proteomics analysis and improve understanding of the WD resistance of plants. Currently, physiological parameters were applied to investigate C. humilis response to WD. Moreover, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in C. humilis leaves subjected to WD (24 d). Furthermore, we also examined the correlation between protein and transcript levels. Several physiological parameters, including relative water content and Pn were reduced by WD. In addition, the malondialdehyde (MDA), relative electrolyte leakage (REL), total soluble sugar, and proline were increased in WD-treated C. humilis . Comparative proteomic analysis revealed 46 protein spots (representing 43 unique proteins) differentially expressed in C. humilis leaves under WD. These proteins were mainly involved in photosynthesis, ROS scavenging, carbohydrate metabolism, transcription, protein synthesis, protein processing, and nitrogen and amino acid metabolisms, respectively. WD promoted the CO 2 assimilation by increase light reaction and Calvin cycle, leading to the reprogramming of carbon metabolism. Moreover, the accumulation of osmolytes (i.e., proline and total soluble sugar) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione s-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under WD. Importantly, the regulation role of carbohydrate metabolisms (e. g. glycolysis, pentose phosphate pathways, and TCA) was enhanced. These findings provide key candidate proteins for genetic improvement of perennial plants metabolism under

Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

Science.gov (United States)

Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

2016-01-01

Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

DEFF Research Database (Denmark)

Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme

2008-01-01

Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats

DEFF Research Database (Denmark)

Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen

2017-01-01

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative...... proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33...... proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb...

Proteomics in studying cancer stem cell biology.

Science.gov (United States)

Kranenburg, Onno; Emmink, Benjamin L; Knol, Jaco; van Houdt, Winan J; Rinkes, Inne H M Borel; Jimenez, Connie R

2012-06-01

Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.

Gel-free proteomics reveal potential biomarkers of priming-induced salt tolerance in durum wheat.

Science.gov (United States)

Fercha, Azzedine; Capriotti, Anna Laura; Caruso, Giuseppe; Cavaliere, Chiara; Gherroucha, Hocine; Samperi, Roberto; Stampachiacchiere, Serena; Lagana, Aldo

2013-10-08

Seed priming has been successfully demonstrated to be an efficient method to improve crop productivity under stressful conditions. As a first step toward better understanding of the mechanisms underlying the priming-induced salt stress tolerance in durum wheat, and to overcome the limitations of the gel-based approach, a comparative gel-free proteomic analysis was conducted with durum wheat seed samples of varying vigor as generated by hydro- and ascorbate-priming treatments. Results indicate that hydro-priming was accompanied by significant changes of 72 proteins, most of which are involved in proteolysis, protein synthesis, metabolism and disease/defense response. Ascorbate-priming was, however, accompanied by significant changes of 83 proteins, which are mainly involved in protein metabolism, antioxidant protection, repair processes and, interestingly, in methionine-related metabolism. The present study provides new information for understanding how 'priming-memory' invokes seed stress tolerance. The current work describes the first study in which gel-free shotgun proteomics were used to investigate the metabolic seed protein fraction in durum wheat. A combined approach of protein fractionation, hydrogel nanoparticle enrichment technique, and gel-free shotgun proteomic analysis allowed us to identify over 380 proteins exhibiting greater molecular weight diversity (ranging from 7 to 258kDa). Accordingly, we propose that this approach could be useful to acquire a wider perspective and a better understanding of the seed proteome. In the present work, we employed this method to investigate the potential biomarkers of priming-induced salt tolerance in durum wheat. In this way, we identified several previously unrecognized proteins which were never been reported before, particularly for the ascorbate-priming treatment. These findings could provide new avenues for improving crop productivity, particularly under unfavorable environmental conditions. © 2013.

Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

Science.gov (United States)

Du, Jing; Guo, Shirong; Sun, Jin; Shu, Sheng

2018-05-01

The mechanism of exogenous Spd-induced Ca(NO 3 ) 2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO 3 ) 2 stress resistance. Ca(NO 3 ) 2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO 3 ) 2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO 3 ) 2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO 3 ) 2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO 3 ) 2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H 2 O 2 and MDA, were detected after exogenous Spd application during Ca(NO 3 ) 2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO 3 ) 2 stress tolerance mediated by exogenous Spd.

Physiological and Proteomics Analyses Reveal Low-Phosphorus Stress Affected the Regulation of Photosynthesis in Soybean.

Science.gov (United States)

Chu, Shanshan; Li, Hongyan; Zhang, Xiangqian; Yu, Kaiye; Chao, Maoni; Han, Suoyi; Zhang, Dan

2018-06-06

Previous studies have revealed a significant genetic relationship between phosphorus (P)-efficiency and photosynthesis-related traits in soybean. In this study, we used proteome profiling in combination with expression analysis, biochemical investigations, and leaf ultrastructural analysis to identify the underlying physiological and molecular responses. The expression analysis and ultrastructural analysis showed that the photosynthesis key genes were decreased at transcript levels and the leaf mesophyll and chloroplast were severely damaged after low-P stress. Approximately 55 protein spots showed changes under low-P condition by mass spectrometry, of which 17 were involved in various photosynthetic processes. Further analysis revealed the depression of photosynthesis caused by low-P stress mainly involves the regulation of leaf structure, adenosine triphosphate (ATP) synthesis, absorption and transportation of CO₂, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. In summary, our findings indicated that the existence of a stringent relationship between P supply and the genomic control of photosynthesis in soybean. As an important strategy to protect soybean photosynthesis, P could maintain the stability of cell structure, up-regulate the enzymes’ activities, recover the process of photosystem II (PSII), and induce the expression of low-P responsive genes and proteins.

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

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Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

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Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-12-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Proteomic analysis of Bifidobacterium longum subsp. infantis reveals the metabolic insight on consumption of prebiotics and host glycans.

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Jae-Han Kim

Full Text Available Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO. To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome

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Marianna Caterino

2018-03-01

Full Text Available Background:/Aims: Renal disease is a common cause of morbidity in patients with Bardet-Biedl syndrome (BBS, however the severity of kidney dysfunction is highly variable. To date, there is little information on the pathogenesis, the risk and predictor factors for poor renal outcome in this setting. The present study aims to analyze the spectrum of urinary proteins in BBS patients, in order to potentially identify 1 disease-specific proteomic profiles that may differentiate the patients from normal subjects; 2 urinary markers of renal dysfunction. Methods: Fourteen individuals (7 males and 7 females with a clinical diagnosis of BBS have been selected in this study. A pool of 10 aged-matched males and 10 aged-matched females have been used as controls for proteomic analysis. The glomerular filtration rate (eGFR has been estimated using the CKD-EPI formula. Variability of eGFR has been retrospectively assessed calculating average annual eGFR decline (ΔeGFR in a mean follow-up period of 4 years (3-7. Results: 42 proteins were significantly over- or under-represented in BBS patients compared with controls; the majority of these proteins are involved in fibrosis, cell adhesion and extracellular matrix organization. Statistic studies revealed a significant correlation between urine fibronectin (u-FN (r2=0.28; p<0.05, CD44 antigen (r2 =0.35; p<0.03 and lysosomal alfa glucosidase ( r20.27; p<0.05 abundance with the eGFR. In addition, u-FN (r2 =0.2389; p<0.05 was significantly correlated with ΔeGFR. Conclusion: The present study demonstrates that urine proteome of BBS patients differs from that of normal subjects; in addition, kidney dysfunction correlated with urine abundance of known markers of renal fibrosis.

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.

Proteomic Studies on Human and Experimental Cerebral Malaria

KAUST Repository

Moussa, Ehab

2012-07-01

Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

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Keller, Lani C; Romijn, Edwin P; Zamora, Ivan; Yates, John R; Marshall, Wallace F

2005-06-21

The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

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Grabek, Katharine R.; Karimpour-Fard, Anis; Epperson, L. Elaine; Hindle, Allyson; Hunter, Lawrence E.

2011-01-01

The hibernator's heart functions continuously and avoids damage across the wide temperature range of winter heterothermy. To define the molecular basis of this phenotype, we quantified proteomic changes in the 13-lined ground squirrel heart among eight distinct physiological states encompassing the hibernator's year. Unsupervised clustering revealed a prominent seasonal separation between the summer homeotherms and winter heterotherms, whereas within-season state separation was limited. Further, animals torpid in the fall were intermediate to summer and winter, consistent with the transitional nature of this phase. A seasonal analysis revealed that the relative abundances of protein spots were mainly winter-increased. The winter-elevated proteins were involved in fatty acid catabolism and protein folding, whereas the winter-depleted proteins included those that degrade branched-chain amino acids. To identify further state-dependent changes, protein spots were re-evaluated with respect to specific physiological state, confirming the predominance of seasonal differences. Additionally, chaperone and heat shock proteins increased in winter, including HSPA4, HSPB6, and HSP90AB1, which have known roles in protecting against ischemia-reperfusion injury and apoptosis. The most significant and greatest fold change observed was a disappearance of phospho-cofilin 2 at low body temperature, likely a strategy to preserve ATP. The robust summer-to-winter seasonal proteomic shift implies that a winter-protected state is orchestrated before prolonged torpor ensues. Additionally, the general preservation of the proteome during winter hibernation and an increase of stress response proteins, together with dephosphorylation of cofilin 2, highlight the importance of ATP-conserving mechanisms for winter cardioprotection. PMID:21914784

Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

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Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

2013-07-11

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion

Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics.

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Kumar, Yashwant; Zhang, Limin; Panigrahi, Priyabrata; Dholakia, Bhushan B; Dewangan, Veena; Chavan, Sachin G; Kunjir, Shrikant M; Wu, Xiangyu; Li, Ning; Rajmohanan, Pattuparambil R; Kadoo, Narendra Y; Giri, Ashok P; Tang, Huiru; Gupta, Vidya S

2016-07-01

Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Clinical proteomic analysis of scrub typhus infection.

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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.

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

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Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

2012-12-01

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

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Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Mapping out starvation responses in yeast by proteomics

DEFF Research Database (Denmark)

Rødkær, Steven Vestergaard; Færgeman, Nils J.; Andersen, Jens S.

2011-01-01

that are involved in this positive outcome. Based on that, processes like autophagy, lipid turnover and the generation/clearance of reactive oxygen species (ROS) have all been describe to affect life span, either alone, or in a not fully characterized interplay. The baker’s yeast Saccharomyces cerevisae is by now...... the organism with the best characterized proteome and is therefore the organism of choice in many proteomic studies. Additionally, this single-celled organism exhibits many conserved proteins and pathways of higher animals, thus observations in the yeast might reveal important information applying to other...

Proteomic and comparative genomic analysis reveals adaptability of Brassica napus to phosphorus-deficient stress.

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Chen, Shuisen; Ding, Guangda; Wang, Zhenhua; Cai, Hongmei; Xu, Fangsen

2015-03-18

Given low solubility and immobility in many soils of the world, phosphorus (P) may be the most widely studied macronutrient for plants. In an attempt to gain an insight into the adaptability of Brassica napus to P deficiency, proteome alterations of roots and leaves in two B. napus contrasting genotypes, P-efficient 'Eyou Changjia' and P-inefficient 'B104-2', under long-term low P stress and short-term P-free starvation conditions were investigated, and proteomic combined with comparative genomic analyses were conducted to interpret the interrelation of differential abundance protein species (DAPs) responding to P deficiency with quantitative trait loci (QTLs) for P deficiency tolerance. P-efficient 'Eyou Changjia' had higher dry weight and P content, and showed high tolerance to low P stress compared with P-inefficient 'B104-2'. A total of 146 DAPs were successfully identified by MALDI TOF/TOF MS, which were categorized into several groups including defense and stress response, carbohydrate and energy metabolism, signaling and regulation, amino acid and fatty acid metabolism, protein process, biogenesis and cellular component, and function unknown. 94 of 146 DAPs were mapped to a linkage map constructed by a B. napus population derived from a cross between the two genotypes, and 72 DAPs were located in the confidence intervals of QTLs for P efficiency related traits. We conclude that the identification of these DAPs and the co-location of DAPs with QTLs in the B. napus linkage genetic map provide us novel information in understanding the adaptability of B. napus to P deficiency, and helpful to isolate P-efficient genes in B. napus. Low P seriously limits the production and quality of B. napus. Proteomics and genetic linkage map were widely used to study the adaptive strategies of B. napus response to P deficiency, proteomic combined with comparative genetic analysis to investigate the correlations between DAPs and QTLs are scarce. Thus, we herein investigated

Differential proteomics reveals the hallmarks of seed development in common bean (Phaseolus vulgaris L.)

NARCIS (Netherlands)

Parreira, J R; Bouraada, J; Fitzpatrick, M A; Silvestre, S; Bernardes da Silva, A; Marques da Silva, J; Almeida, A M; Fevereiro, P; Altelaar, A F M; Araújo, S S

2016-01-01

Common bean (Phaseolus vulgaris L.) is one of the most consumed staple foods worldwide. Little is known about the molecular mechanisms controlling seed development. This study aims to comprehensively describe proteome dynamics during seed development of common bean. A high-throughput gel-free

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

Science.gov (United States)

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.

Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis.

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Guo, Zhiling; Hu, Qin; Tian, Jijing; Yan, Li; Jing, Chuanyong; Xie, Heidi Qunhui; Bao, Wenjun; Rice, Robert H; Zhao, Bin; Jiang, Guibin

2016-11-01

Proteomics technology is an attractive biomarker candidate discovery tool that can be applied to study large sets of biological molecules. To identify novel biomarkers and molecular targets in arsenic-induced skin lesions, we have determined the protein profile of arsenic-affected human epidermal stratum corneum by shotgun proteomics. Samples of palm and foot sole from healthy subjects were analyzed, demonstrating similar protein patterns in palm and sole. Samples were collected from the palms of subjects with arsenic keratosis (lesional and adjacent non-lesional samples) and arsenic-exposed subjects without lesions (normal). Samples from non-exposed healthy individuals served as controls. We found that three proteins in arsenic-exposed lesional epidermis were consistently distinguishably expressed from the unaffected epidermis. One of these proteins, the cadherin-like transmembrane glycoprotein, desmoglein 1 (DSG1) was suppressed. Down-regulation of DSG1 may lead to reduced cell-cell adhesion, resulting in abnormal epidermal differentiation. The expression of keratin 6c (KRT6C) and fatty acid binding protein 5 (FABP5) were significantly increased. FABP5 is an intracellular lipid chaperone that plays an essential role in fatty acid metabolism in human skin. This raises a possibility that overexpression of FABP5 may affect the proliferation or differentiation of keratinocytes by altering lipid metabolism. KRT6C is a constituent of the cytoskeleton that maintains epidermal integrity and cohesion. Abnormal expression of KRT6C may affect its structural role in the epidermis. Our findings suggest an important approach for future studies of arsenic-mediated toxicity and skin cancer, where certain proteins may represent useful biomarkers of early diagnoses in high-risk populations and hopefully new treatment targets. Further studies are required to understand the biological role of these markers in skin pathogenesis from arsenic exposure. Copyright © 2016 Elsevier Ltd

The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats

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Xu Yu-Dong

2010-08-01

Full Text Available Abstract Background The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR, which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma. Methods In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism. Results In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses. Conclusions Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

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Yener Bülent

2007-10-01

Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

Calorimetric monitoring of the serum proteome in schizophrenia patients

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Krumova, Sashka [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Rukova, Blaga [Department of Medical Genetics, Medical University of Sofia, 2 Zdrave Str., Sofia 1431 (Bulgaria); Todinova, Svetla [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Gartcheva, Lidia [National Specialized Hospital for Active Treating of Haematological Diseases, 6 Plovdivsko pole Str., Sofia 1756 (Bulgaria); Milanova, Vihra [Department of Psychiatry, Medical University of Sofia, 1 Sv. Georgi Sofiiski Str., Sofia 1431 (Bulgaria); Toncheva, Draga [Department of Medical Genetics, Medical University of Sofia, 2 Zdrave Str., Sofia 1431 (Bulgaria); Taneva, Stefka G., E-mail: stefka.germanova@ehu.es [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria)

2013-11-20

Highlights: • DSC reveals modified thermal behavior of blood serum from schizophrenic patients. • The high-abundance portion of the serum proteome is thermally stabilized in Sz. • The Sz plasma thermograms are classified in four distinct calorimetric groups. • The effectiveness of drug treatment correlates with the plasma thermodynamic behavior. - Abstract: Schizophrenia (Sz) is a multifactorial mental disorder with high frequency. Due to its chronic and relapsing nature there is a strong need for biomarkers for early psychosis detection and objective evaluation of drug (usually antipsychotics) treatment effect. Here differential scanning calorimetry (DSC) is applied to thermodynamically characterize the blood serum proteome of paranoid schizophrenia patients on routine antipsychotic treatment in comparison to healthy controls. DSC revealed significant modifications in the thermodynamic behavior of blood sera from Sz patients, the overall thermal profile being changed in all Sz cases under study. The calorimetric profiles were classified in four distinct groups, reflecting different thermal stabilization of the high-abundance portion of the serum proteome. The observed positive (thermograms becoming closer to the healthy profile) or negative (thermograms deviating stronger from the healthy profile) proteome thermal stability switches and the Sz thermograms persistence in patients’ follow-up corresponded well with the effect of drug treatment.

Calorimetric monitoring of the serum proteome in schizophrenia patients

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Krumova, Sashka; Rukova, Blaga; Todinova, Svetla; Gartcheva, Lidia; Milanova, Vihra; Toncheva, Draga; Taneva, Stefka G.

2013-01-01

Highlights: • DSC reveals modified thermal behavior of blood serum from schizophrenic patients. • The high-abundance portion of the serum proteome is thermally stabilized in Sz. • The Sz plasma thermograms are classified in four distinct calorimetric groups. • The effectiveness of drug treatment correlates with the plasma thermodynamic behavior. - Abstract: Schizophrenia (Sz) is a multifactorial mental disorder with high frequency. Due to its chronic and relapsing nature there is a strong need for biomarkers for early psychosis detection and objective evaluation of drug (usually antipsychotics) treatment effect. Here differential scanning calorimetry (DSC) is applied to thermodynamically characterize the blood serum proteome of paranoid schizophrenia patients on routine antipsychotic treatment in comparison to healthy controls. DSC revealed significant modifications in the thermodynamic behavior of blood sera from Sz patients, the overall thermal profile being changed in all Sz cases under study. The calorimetric profiles were classified in four distinct groups, reflecting different thermal stabilization of the high-abundance portion of the serum proteome. The observed positive (thermograms becoming closer to the healthy profile) or negative (thermograms deviating stronger from the healthy profile) proteome thermal stability switches and the Sz thermograms persistence in patients’ follow-up corresponded well with the effect of drug treatment

Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

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Soman, Kizhake V; Stafford, Susan J; Pazdrak, Konrad; Wu, Zheng; Luo, Xuemei; White, Wendy I; Wiktorowicz, John E; Calhoun, William J; Kurosky, Alexander

2017-08-04

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

Characterization of the canine urinary proteome.

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Brandt, Laura E; Ehrhart, E J; Scherman, Hataichanok; Olver, Christine S; Bohn, Andrea A; Prenni, Jessica E

2014-06-01

Urine is an attractive biofluid for biomarker discovery as it is easy and minimally invasive to obtain. While numerous studies have focused on the characterization of human urine, much less research has focused on canine urine. The objectives of this study were to characterize the universal canine urinary proteome (both soluble and exosomal), to determine the overlap between the canine proteome and a representative human urinary proteome study, to generate a resource for future canine studies, and to determine the suitability of the dog as a large animal model for human diseases. The soluble and exosomal fractions of normal canine urine were characterized using liquid chromatography tandem mass spectrometry (LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was utilized to assign the canine urinary proteome to respective Gene Ontology categories, such as Cellular Component, Molecular Function, and Biological Process. Over 500 proteins were confidently identified in normal canine urine. Gene Ontology analysis revealed that exosomal proteins were largely derived from an intracellular location, while soluble proteins included both extracellular and membrane proteins. Exosome proteins were assigned to metabolic processes and localization, while soluble proteins were primarily annotated to specific localization processes. Several proteins identified in normal canine urine have previously been identified in human urine where these proteins are related to various extrarenal and renal diseases. The results of this study illustrate the potential of the dog as an animal model for human disease states and provide the framework for future studies of canine renal diseases. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

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Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

2015-01-01

Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

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García-Berrocoso, Teresa; Llombart, Víctor; Colàs-Campàs, Laura; Hainard, Alexandre; Licker, Virginie; Penalba, Anna; Ramiro, Laura; Simats, Alba; Bustamante, Alejandro; Martínez-Saez, Elena; Canals, Francesc; Sanchez, Jean-Charles; Montaner, Joan

2018-01-01

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets. © 2018 by The American Society for

Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

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Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

2014-12-05

Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

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.

Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

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Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

2017-03-01

In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of Proteomics to the Study of Pollination Drops

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Natalie Prior

2013-04-01

Full Text Available Premise of the study: Pollination drops are a formative component in gymnosperm pollen-ovule interactions. Proteomics offers a direct method for the discovery of proteins associated with this early stage of sexual reproduction. Methods: Pollination drops were sampled from eight gymnosperm species: Chamaecyparis lawsoniana (Port Orford cedar, Ephedra monosperma, Ginkgo biloba, Juniperus oxycedrus (prickly juniper, Larix ×marschlinsii, Pseudotsuga menziesii (Douglas-fir, Taxus ×media, and Welwitschia mirabilis. Drops were collected by micropipette using techniques focused on preventing sample contamination. Drop proteins were separated using both gel and gel-free methods. Tandem mass spectrometric methods were used including a triple quadrupole and an Orbitrap. Results: Proteins are present in all pollination drops. Consistency in the protein complement over time was shown in L. ×marschlinsii. Representative mass spectra from W. mirabilis chitinase peptide and E. monosperma serine carboxypeptidase peptide demonstrated high quality results. We provide a summary of gymnosperm pollination drop proteins that have been discovered to date via proteomics. Discussion: Using proteomic methods, a dozen classes of proteins have been identified to date. Proteomics presents a way forward in deepening our understanding of the biological function of pollination drops.

Salinity-induced inhibition of growth in the aquatic pteridophyte Azolla microphylla primarily involves inhibition of photosynthetic components and signaling molecules as revealed by proteome analysis.

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Thagela, Preeti; Yadav, Ravindra Kumar; Mishra, Vagish; Dahuja, Anil; Ahmad, Altaf; Singh, Pawan Kumar; Tiwari, Budhi Sagar; Abraham, Gerard

2017-01-01

Salinity stress causes adverse physiological and biochemical changes in the growth and productivity of a plant. Azolla, a symbiotic pteridophyte and potent candidate for biofertilizer due to its nitrogen fixation ability, shows reduced growth and nitrogen fixation during saline stress. To better understand regulatory components involved in salinity-induced physiological changes, in the present study, Azolla microphylla plants were exposed to NaCl (6.74 and 8.61 ds/m) and growth, photochemical reactions of photosynthesis, ion accumulation, and changes in cellular proteome were studied. Maximum dry weight was accumulated in control and untreated plant while a substantial decrease in dry weight was observed in the plants exposed to salinity. Exposure of the organism to different concentrations of salt in hydroponic conditions resulted in differential level of Na + and K + ion accumulation. Comparative analysis of salinity-induced proteome changes in A. microphylla revealed 58 salt responsive proteins which were differentially expressed during the salt exposure. Moreover, 42 % spots among differentially expressed proteins were involved in different signaling events. The identified proteins are involved in photosynthesis, energy metabolism, amino acid biosynthesis, protein synthesis, and defense. Downregulation of these key metabolic proteins appears to inhibit the growth of A. microphylla in response to salinity. Altogether, the study revealed that in Azolla, increased salinity primarily affected signaling and photosynthesis that in turn leads to reduced biomass.

Toxicological effects of benzo(a)pyrene, DDT and their mixture on the green mussel Perna viridis revealed by proteomic and metabolomic approaches.

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Song, Qinqin; Chen, Hao; Li, Yuhu; Zhou, Hailong; Han, Qian; Diao, Xiaoping

2016-02-01

Benzo(a)pyrene (BaP) and dichlorodiphenyltrichloroethane (DDT) are persistent organic pollutants and environmental estrogens (EEs) with known toxicity towards the green mussel, Perna viridis. In this study, the toxic effects of BaP (10 µg/L) and DDT (10 µg/L) and their mixture were assessed in green mussel gills with proteomic and metabolomic approaches. Metabolic responses indicated that BaP mainly caused disturbance in osmotic regulation by significantly decrease in branched chain amino acids, dimethylamine and dimethylglycine in gills of male green mussels after exposure for 7 days. DDT mainly caused disturbance in osmotic regulation and energy metabolism by differential alteration of betaine, dimethylamine, dimethylglycine, amino acids, and succinate in gills of male green mussels. However, the mixture of BaP and DDT didn't show obvious metabolite changes. Proteomic analysis showed different protein expression profiles between different treatment groups, which demonstrated that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced cell apoptosis, disturbance in protein digestion and energy metabolism in gills of green mussels, whereas DDT exposure altered proteins that were associated with oxidative stress, cytoskeleton and cell structure, protein digestion and energy metabolism. However, the mixture of BaP and DDT affected proteins related to the oxidative stress, cytoskeleton and cell structure, protein biosynthesis and modification, energy metabolism, growth and apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

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Susann Kummer

Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

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].

Proteomic analysis reveals changes in carbohydrate and protein metabolism associated with broiler breast myopathy.

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Kuttappan, Vivek A; Bottje, Walter; Ramnathan, Ranjith; Hartson, Steven D; Coon, Craig N; Kong, Byung-Whi; Owens, Casey M; Vazquez-Añon, Mercedes; Hargis, Billy M

2017-08-01

White Striping (WS) and Woody Breast (WB) are 2 conditions that adversely affect consumer acceptance as well as quality of poultry meat and meat products. Both WS and WB are characterized with degenerative myopathic changes. Previous studies showed that WS and WB in broiler fillets could result in higher ultimate pH, increased drip loss, and decreased marinade uptake. The main objective of the present study was to compare the proteomic profiles of muscle tissue (n = 5 per group) with either NORM (no or few minor myopathic lesions) or SEV (with severe myopathic changes). Proteins were extracted from these samples and analyzed using a hybrid LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). Over 800 proteins were identified in the muscle samples, among which 141 demonstrated differential (P < 0.05) expression between NORM and SEV. The set of differentially (P < 0.05) expressed proteins was uploaded to Ingenuity Pathway Analysis® (IPA) software to determine the associated biological networks and pathways. The IPA analysis showed that eukaryotic initiation factor-2 (eIF-2) signaling, mechanistic target of rapamycin (mTOR) signaling, as well as regulation of eIF4 and p70S6K signaling were the major canonical pathways up-regulated (P < 0.05) in SEV muscle compared to NORM. The up-regulation of these pathways indicate an increase in protein synthesis which could be part of the rapid growth as well as cellular stress associated with ongoing muscle degeneration and the attempt to repair tissue damage in SEV birds. Furthermore, IPA analysis revealed that glycolysis and gluconeogenesis were the major down-regulated (P < 0.05) canonical pathways in SEV with respect to NORM muscle. Down-regulation of these pathways could be the reason for higher ultimate pH seen in SEV muscle samples indicating reduced glycolytic potential. In conclusion, comparison of proteomic profiles of NORM and SEV muscle samples showed differences in protein profile which explains some of the observed

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

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Panga Jaipal Reddy

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

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

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Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection

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National Aeronautics and Space Administration — To reveal outcomes of microgravity on molecular processes within the cellular environment we have employed a mass-spectrometry based proteomics approach. Proteomics...

Proteomics Coupled with Metabolite and Cell Wall Profiling Reveal Metabolic Processes of a Developing Rice Stem Internode

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Lin, Fan; Williams, Brad J.; Thangella, Padmavathi A. V.; Ladak, Adam; Schepmoes, Athena A.; Olivos, Hernando J.; Zhao, Kangmei; Callister, Stephen J.; Bartley, Laura E.

2017-07-13

Internodes of grass stems function in mechanical support, transport, and, in some species, are a major sink organ for carbon in the form of cell wall polymers. This study reports cell wall composition, proteomic and metabolite analyses of the rice elongating internode. Along eight segments of the second rice internode (internode II) at booting stage, cellulose, lignin, and xylose increase as a percentage of cell wall material from the younger to the older internode segments, indicating active cell wall synthesis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) of trypsin-digested peptides of size-fractionated proteins extracted from this internode at booting reveals 2547proteins with at least two unique peptides. The dataset includes many glycosyltransferases, acyltransferases, glycosyl hydrolases, cell wall-localized proteins, and protein kinases that have or may have functions in cell wall biosynthesis or remodeling. Phospho-enrichment of the internode II peptides identified 21 unique phosphopeptides belonging to 20 phosphoproteins including an LRR-III family receptor like kinase. GO over-representation and KEGG pathway analyses highlight the abundances of internode proteins involved in biosynthetic processes, especially the synthesis of secondary metabolites such as phenylpropanoids and flavonoids. LC-MS of hot methanol-extracted secondary metabolites from internode II at four stages (elongation, early mature, mature and post mature) indicates that secondary metabolites in stems are distinct from those of roots and leaves, and differ during stem maturation. This work fills a void of knowledge of proteomics and metabolomics data for grass stems, specifically for rice, and provides baseline knowledge for more detailed studies of cell wall synthesis and other biological processes during internode development, toward improving grass agronomic properties.

Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

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Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

2013-04-01

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic explorations of autism spectrum disorder.

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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.

Proteome regulation during Olea europaea fruit development.

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Linda Bianco

Full Text Available Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes.In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies.This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Proteome regulation during Olea europaea fruit development.

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Bianco, Linda; Alagna, Fiammetta; Baldoni, Luciana; Finnie, Christine; Svensson, Birte; Perrotta, Gaetano

2013-01-01

Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Integrating cell biology and proteomic approaches in plants.

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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

[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.

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

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Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

2017-01-01

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

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Dominik A. Megger

2017-09-01

Full Text Available Interferons (IFNs are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction. In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

Differential proteomic analysis reveals sequential heat stress-responsive regulatory network in radish (Raphanus sativus L.) taproot.

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Wang, Ronghua; Mei, Yi; Xu, Liang; Zhu, Xianwen; Wang, Yan; Guo, Jun; Liu, Liwang

2018-05-01

Differential abundance protein species (DAPS) involved in reducing damage and enhancing thermotolerance in radish were firstly identified. Proteomic analysis and omics association analysis revealed a HS-responsive regulatory network in radish. Heat stress (HS) is a major destructive factor influencing radish production and supply in summer, for radish is a cool season vegetable crop being susceptible to high temperature. In this study, the proteome changes of radish taproots under 40 °C treatment at 0 h (Control), 12 h (Heat12) and 24 h (Heat24) were analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) approach. In total, 2258 DAPS representing 1542 differentially accumulated uniprotein species which respond to HS were identified. A total of 604, 910 and 744 DAPS was detected in comparison of Control vs. Heat12, Control vs. Heat24, and Heat12 vs. Heat24, respectively. Gene ontology and pathway analysis showed that annexin, ubiquitin-conjugating enzyme, ATP synthase, heat shock protein (HSP) and other stress-related proteins were predominately enriched in signal transduction, stress and defense pathways, photosynthesis and energy metabolic pathways, working cooperatively to reduce stress-induced damage in radish. Based on iTRAQ combined with the transcriptomics analysis, a schematic model of a sequential HS-responsive regulatory network was proposed. The initial sensing of HS occurred at the plasma membrane, and then key components of stress signal transduction triggered heat-responsive genes in the plant protective metabolism to re-establish homeostasis and enhance thermotolerance. These results provide new insights into characteristics of HS-responsive DAPS and facilitate dissecting the molecular mechanisms underlying heat tolerance in radish and other root crops.

Strain-resolved microbial community proteomics reveals simultaneous aerobic and anaerobic function during gastrointestinal tract colonization of a preterm infant

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Brandon eBrooks

2015-07-01

Full Text Available While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13-21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains of Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.

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

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

2016-11-04

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

Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction

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Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing

2016-11-11

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies

Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus

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Ann Ray

2016-07-01

Full Text Available Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells.

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

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Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-01-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

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Aayudh Das

2016-01-01

Full Text Available Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

Proteomic Analysis Reveals the Deregulation of Inflammation-Related Proteins in Acupuncture-Treated Rats with Asthma Onset

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Yu-Dong Xu

2012-01-01

Full Text Available Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE and mass-spectrometry- (MS- based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11 and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE. These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

KAUST Repository

Dineshram, R.

2015-10-28

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world\\'s edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

KAUST Repository

Dineshram, R.; Q., Quan; Sharma, Rakesh; Chandramouli, Kondethimmanahalli; Yalamanchili, Hari Krishna; Chu, Ivan; Thiyagarajan, Vengatesen

2015-01-01

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

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.

Proteomic Profiles Reveal the Function of Different Vegetative Tissues of Moringa oleifera.

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Wang, Lei; Zou, Qiong; Wang, Jinxing; Zhang, Junjie; Liu, Zeping; Chen, Xiaoyang

2016-12-01

Moringa oleifera is a rich source of bioactive compounds and is widely used in traditional medicine and food for its nutritional value; however, the protein and peptide components of different tissues are rarely discussed. Here, we describe the first investigation of M. oleifera proteomes using mass spectrometry and bioinformatics methods. We aimed to elucidate the protein profiles of M. oleifera leaves, stem, bark, and root. Totally 202 proteins were identified from four vegetative organs. We identified 101 proteins from leaves, 51 from stem, 94 from bark and 67 from root, finding that only five proteins existed in both four vegetative parts. The calculated pI of most of the proteins is distributed in 5-10 and the molecular weight distributed below 100 kDa. Functional classification analysis revealed that proteins which are involved in catalytic activities are the most abundant both in leaves, stem, bark and root. Identification of several heat shock proteins in four vegetative tissues might be adaptive for resistance to high temperature environmental stresses of tropical or subtropical areas. Some enzymes involved in antioxidant processes were also identified in M. oleifera leaves, stem, bark and root. Among the four tissues studies here, leaves protein content and molecular diversity were the highest. The identification of the flocculating protein MO2.1 and MO2.2 in the bark and root provides clue to clarify the antimicrobial molecular mechanisms of root and bark. This study provides information on the protein compositions of M. oleifera vegetative tissues that will be beneficial for potential drug and food supplement development and plant physiology research.

Proteomic identification of gender molecular markers in Bothrops jararaca venom.

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

2016-04-29

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

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.

Physiological and comparative proteomic analysis reveals different drought responses in roots and leaves of drought-tolerant wild wheat (Triticum boeoticum.

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Hui Liu

Full Text Available To determine the proteomic-level responses of drought tolerant wild wheat (Triticum boeoticum, physiological and comparative proteomic analyses were conducted using the roots and the leaves of control and short term drought-stressed plants. Drought stress was imposed by transferring hydroponically grown seedlings at the 3-leaf stage into 1/2 Hoagland solution containing 20% PEG-6000 for 48 h. Root and leaf samples were separately collected at 0 (control, 24, and 48 h of drought treatment for analysis. Physiological analysis indicated that abscisic acid (ABA level was greatly increased in the drought-treated plants, but the increase was greater and more rapid in the leaves than in the roots. The net photosynthetic rate of the wild wheat leaves was significantly decreased under short-term drought stress. The deleterious effects of drought on the studied traits mainly targeted photosynthesis. Comparative proteomic analysis identified 98 and 85 differently changed protein spots (DEPs (corresponding to 87 and 80 unique proteins, respectively in the leaves and the roots, respectively, with only 6 mutual unique proteins in the both organs. An impressive 86% of the DEPs were implicated in detoxification and defense, carbon metabolism, amino acid and nitrogen metabolism, proteins metabolism, chaperones, transcription and translation, photosynthesis, nucleotide metabolism, and signal transduction. Further analysis revealed some mutual and tissue-specific responses to short-term drought in the leaves and the roots. The differences of drought-response between the roots and the leaves mainly included that signal sensing and transduction-associated proteins were greatly up-regulated in the roots. Photosynthesis and carbon fixation ability were decreased in the leaves. Glycolysis was down-regulated but PPP pathway enhanced in the roots, resulting in occurrence of complex changes in energy metabolism and establishment of a new homeostasis. Protein metabolism

A review of studies of the proteomes of circulating microparticles

DEFF Research Database (Denmark)

Nielsen, Christoffer Tandrup; Østergaard, Ole; Rasmussen, Niclas S

2017-01-01

of the specific proteins and their quantities, i.e. the proteome, in complex samples such as MPs enables an in-depth characterization of the phenotypical changes of the MPs during disease states. At present, only a limited number of proteomic studies of circulating MPs have been carried out in healthy individuals...... and disease populations. Interestingly, these studies indicate that a small set of MP-proteins, in particular, overexpression of galectin-3-binding protein (G3BP) distinguish MPs in patients with venous thromboembolism and the systemic autoimmune disease, systemic lupus erythematosus (SLE). G3BP is important...

Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.

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Maria Liguori

Full Text Available The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF proteomic profiles of Multiple Sclerosis (MS patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS, 16 Relapsing Remitting (RR MS, 11 Progressive (Pr MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000-25000 Da. Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥ 1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05, whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04. Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013. Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS.

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

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Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

2018-04-10

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

Proteomics of Maize Root Development.

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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

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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.

Nucleophosmin in the pathogenesis of arsenic-related bladder carcinogenesis revealed by quantitative proteomics

International Nuclear Information System (INIS)

Chen Shuhui; Wang Yiwen; Hsu Jueliang; Chang Hongyi; Wang Chiyun; Shen Potsun; Chiang Chiwu; Chuang Jingjing; Tsai Hungwen; Gu Powen; Chang Fangchih; Liu Hsiaosheng; Chow Nanhaw

2010-01-01

To investigate the molecular mechanisms of arsenic (As)-associated carcinogenesis, we performed proteomic analysis on E7 immortalized human uroepithelial cells after treatment with As in vitro. Quantitative proteomics was performed using stable isotope dimethyl labeling coupled with two-dimensional liquid chromatography peptide separation and mass spectrometry (MS)/MS analysis. Among 285 proteins, a total of 26 proteins were upregulated (ratio > 2.0) and 18 proteins were downregulated (ratio < 0.65) by As treatment, which are related to nucleotide binding, lipid metabolism, protein folding, protein biosynthesis, transcription, DNA repair, cell cycle control, and signal transduction. This study reports the potential significance of nucleophosmin (NPM) in the As-related bladder carcinogenesis. NPM was universally expressed in all of uroepithelial cell lines examined, implying that NPM may play a role in human bladder carcinogenesis. Upregulation of NPM tends to be dose- and time-dependent after As treatment. Expression of NPM was associated with cell proliferation, migration and anti-apoptosis. On the contrary, soy isoflavones inhibited the expression of NPM in vitro. The results suggest that NPM may play a role in the As-related bladder carcinogenesis, and soybean-based foods may have potential in the suppression of As/NPM-related tumorigenesis.

Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats.

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Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

2017-10-01

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

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Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

2016-01-15

Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

A label-free quantitative shotgun proteomics analysis of rice grain development

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Koh Hee-Jong

2011-09-01

Full Text Available Abstract Background Although a great deal of rice proteomic research has been conducted, there are relatively few studies specifically addressing the rice grain proteome. The existing rice grain proteomic researches have focused on the identification of differentially expressed proteins or monitoring protein expression patterns during grain filling stages. Results Proteins were extracted from rice grains 10, 20, and 30 days after flowering, as well as from fully mature grains. By merging all of the identified proteins in this study, we identified 4,172 non-redundant proteins with a wide range of molecular weights (from 5.2 kDa to 611 kDa and pI values (from pH 2.9 to pH 12.6. A Genome Ontology category enrichment analysis for the 4,172 proteins revealed that 52 categories were enriched, including the carbohydrate metabolic process, transport, localization, lipid metabolic process, and secondary metabolic process. The relative abundances of the 1,784 reproducibly identified proteins were compared to detect 484 differentially expressed proteins during rice grain development. Clustering analysis and Genome Ontology category enrichment analysis revealed that proteins involved in the metabolic process were enriched through all stages of development, suggesting that proteome changes occurred even in the desiccation phase. Interestingly, enrichments of proteins involved in protein folding were detected in the desiccation phase and in fully mature grain. Conclusion This is the first report conducting comprehensive identification of rice grain proteins. With a label free shotgun proteomic approach, we identified large number of rice grain proteins and compared the expression patterns of reproducibly identified proteins during rice grain development. Clustering analysis, Genome Ontology category enrichment analysis, and the analysis of composite expression profiles revealed dynamic changes of metabolisms during rice grain development. Interestingly, we

Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

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Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

2016-12-01

During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

Functional proteomics of barley and barley chloroplasts – strategies, methods and perspectives

DEFF Research Database (Denmark)

Petersen, Jørgen; Rogowska-Wrzesinska, Adelina; Jensen, Ole Nørregaard

2013-01-01

Barley (Hordeum vulgare) is an important cereal grain that is used in a range of products for animal and human consumption. Crop yield and seed quality has been optimized during decades by plant breeding programs supported by biotechnology and molecular biology techniques. The recently completed...... whole-genome sequencing of barley revealed approximately 26,100 open reading frames, which provides a foundation for detailed molecular studies of barley by functional genomics and proteomics approaches. Such studies will provide further insights into the mechanisms of, for example, drought and stress...... tolerance, micronutrient utilization, and photosynthesis in barley. In the present review we present the current state of proteomics research for investigations of barley chloroplasts, i.e., the organelle that contain the photosynthetic apparatus in the plant. We describe several different proteomics...

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...

Proteomics approaches shed new light on hibernation physiology.

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Grabek, Katharine R; Martin, Sandra L; Hindle, Allyson G

2015-08-01

The broad phylogenetic distribution and rapid phenotypic transitions of mammalian hibernators imply that hibernation is accomplished by differential expression of common genes. Traditional candidate gene approaches have thus far explained little of the molecular mechanisms underlying hibernation, likely due to (1) incomplete and imprecise sampling of a complex phenotype, and (2) the forming of hypotheses about which genes might be important based on studies of model organisms incapable of such dynamic physiology. Unbiased screening approaches, such as proteomics, offer an alternative means to discover the cellular underpinnings that permit successful hibernation and may reveal previously overlooked, important pathways. Here, we review the findings that have emerged from proteomics studies of hibernation. One striking feature is the stability of the proteome, especially across the extreme physiological shifts of torpor-arousal cycles during hibernation. This has led to subsequent investigations of the role of post-translational protein modifications in altering protein activity without energetically wasteful removal and rebuilding of protein pools. Another unexpected finding is the paucity of universal proteomic adjustments across organ systems in response to the extreme metabolic fluctuations despite the universality of their physiological challenges; rather each organ appears to respond in a unique, tissue-specific manner. Additional research is needed to extend and synthesize these results before it will be possible to address the whole body physiology of hibernation.

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

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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.

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

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Molina, Laurence; Salvetat, Nicolas; Ameur, Randa Ben; Peres, Sabine; Sommerer, Nicolas; Jarraya, Fayçal; Ayadi, Hammadi; Molina, Franck; Granier, Claude

2011-12-10

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Contribution of proteomics to the study of plant pathogenic fungi.

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Gonzalez-Fernandez, Raquel; Jorrin-Novo, Jesus V

2012-01-01

Phytopathogenic fungi are one of the most damaging plant parasitic organisms, and can cause serious diseases and important yield losses in crops. The study of the biology of these microorganisms and the interaction with their hosts has experienced great advances in recent years due to the development of moderm, holistic and high-throughput -omic techniques, together with the increasing number of genome sequencing projects and the development of mutants and reverse genetics tools. We highlight among these -omic techniques the importance of proteomics, which has become a relevant tool in plant-fungus pathosystem research. Proteomics intends to identify gene products with a key role in pathogenicity and virulence. These studies would help in the search of key protein targets and in the development of agrochemicals, which may open new ways for crop disease diagnosis and protection. In this review, we made an overview on the contribution of proteomics to the knowledge of life cycle, infection mechanisms, and virulence of the plant pathogenic fungi. Data from current, innovative literature, according to both methodological and experimental systems, were summarized and discussed. Specific sections were devoted to the most studied fungal phytopathogens: Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum.

Proteome Analysis Reveals Extensive Light Stress-Response Reprogramming in the Seagrass Zostera muelleri (Alismatales, Zosteraceae) Metabolism.

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Kumar, Manoj; Padula, Matthew P; Davey, Peter; Pernice, Mathieu; Jiang, Zhijian; Sablok, Gaurav; Contreras-Porcia, Loretto; Ralph, Peter J

2016-01-01

Seagrasses are marine ecosystem engineers that are currently declining in abundance at an alarming rate due to both natural and anthropogenic disturbances in ecological niches. Despite reports on the morphological and physiological adaptations of seagrasses to extreme environments, little is known of the molecular mechanisms underlying photo-acclimation, and/or tolerance in these marine plants. This study applies the two-dimensional isoelectric focusing (2D-IEF) proteomics approach to identify photo-acclimation/tolerance proteins in the marine seagrass Zostera muelleri . For this, Z. muelleri was exposed for 10 days in laboratory mesocosms to saturating (control, 200 μmol photons m -2 s -1 ), super-saturating (SSL, 600 μmol photons m -2 s -1 ), and limited light (LL, 20 μmol photons m -2 s -1 ) irradiance conditions. Using LC-MS/MS analysis, 93 and 40 protein spots were differentially regulated under SSL and LL conditions, respectively, when compared to the control. In contrast to the LL condition, Z. muelleri robustly tolerated super-saturation light than control conditions, evidenced by their higher relative maximum electron transport rate and minimum saturating irradiance values. Proteomic analyses revealed up-regulation and/or appearances of proteins belonging to the Calvin-Benson and Krebs cycle, glycolysis, the glycine cleavage system of photorespiration, and the antioxidant system. These proteins, together with those from the inter-connected glutamate-proline-GABA pathway, shaped Z. muelleri photosynthesis and growth under SSL conditions. In contrast, the LL condition negatively impacted the metabolic activities of Z. muelleri by down-regulating key metabolic enzymes for photosynthesis and the metabolism of carbohydrates and amino acids, which is consistent with the observation with lower photosynthetic performance under LL condition. This study provides novel insights into the underlying molecular photo-acclimation mechanisms in Z. muelleri , in addition

Proteomics of red and white corolla limbs in petunia reveals a novel function of the anthocyanin regulator ANTHOCYANIN1 in determining flower longevity.

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Prinsi, Bhakti; Negri, Alfredo S; Quattrocchio, Francesca M; Koes, Ronald E; Espen, Luca

2016-01-10

The Petunia hybrida ANTHOCYANIN1 (AN1) gene encodes a transcription factor that regulates both the expression of genes involved in anthocyanin synthesis and the acidification of the vacuolar lumen in corolla epidermal cells. In this work, the comparison between the red flowers of the R27 line with the white flowers of the isogenic an1 mutant line W225 showed that the AN1 gene has further pleiotropic effects on flavonoid biosynthesis as well as on distant physiological traits. The proteomic profiling showed that the an1 mutation was associated to changes in accumulation of several proteins, affecting both anthocyanin synthesis and primary metabolism. The flavonoid composition study confirmed that the an1 mutation provoked a broad attenuation of the entire flavonoid pathway, probably by indirect biochemical events. Moreover, proteomic changes and variation of biochemical parameters revealed that the an1 mutation induced a delay in the onset of flower senescence in W225, as supported by the enhanced longevity of the W225 flowers in planta and the loss of sensitivity of cut flowers to sugar. This study suggests that AN1 is possibly involved in the perception and/or transduction of ethylene signal during flower senescence. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic analysis reveals metabolic and regulatory systems involved the syntrophic and axenic lifestyle of Syntrophomonas wolfei.

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Jessica Rhea Sieber

2015-02-01

Full Text Available Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomic approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis macromolecular stability and energy metabolism to S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits.

Science.gov (United States)

Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin

2014-06-13

Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

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,

Data from proteome analysis of Lasiodiplodia theobromae (Botryosphaeriaceae

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Carla C. Uranga

2017-08-01

Full Text Available Trunk disease fungi are a global problem affecting many economically important fruiting trees. The Botryosphaeriaceae are a family of trunk disease fungi that require detailed biochemical characterization in order to gain insight into their pathogenicity. The application of a modified Folch extraction to protein extraction from the Botryosphaeriaceae Lasiodiplodia theobromae generated an unprecedented data set of protein identifications from fragmentation analysis and de novo peptide sequencing of its proteome. This article contains data from protein identifications obtained from a database-dependent fragmentation analysis using three different proteomics algorithms (MSGF, Comet and X! Tandem via the SearchGUI proteomics pipeline program and de novo peptide sequencing. Included are data sets of gene ontology annotations using an all-Uniprot ontology database, as well as a Saccharomyces cerevisiae-only and a Candida albicans-only ontology database, in order to discern between those proteins involved in common functions with S. cerevisiae and those in common with the pathogenic yeast C. albicans. Our results reveal the proteome of L. theobromae contains more ontological categories in common to C. albicans, yet possesses a much wider metabolic repertoire than any of the yeasts studied in this work. Many novel proteins of interest were identified for further biochemical characterization and annotation efforts, as further discussed in the article referencing this article (1. Interactive Cytoscape networks of molecular functions of identified peptides using an all-Uniprot ontological database are included. Data, including raw data, are available via ProteomeXchange with identifier PXD005283.

Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals.

Science.gov (United States)

Himaya, S W A; Marí, Frank; Lewis, Richard J

2018-01-10

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

Proteomics of Trypanosoma evansi infection in rodents.

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Roy, Nainita; Nageshan, Rishi Kumar; Pallavi, Rani; Chakravarthy, Harshini; Chandran, Syama; Kumar, Rajender; Gupta, Ashok Kumar; Singh, Raj Kumar; Yadav, Suresh Chandra; Tatu, Utpal

2010-03-22

Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS). Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more. Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the

Proteomics of Trypanosoma evansi infection in rodents.

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Nainita Roy

2010-03-01

Full Text Available Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS.Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta.

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Kui Shi

Full Text Available Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs and proteins (DEPs, respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

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

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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/

In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics

DEFF Research Database (Denmark)

Adachi, Jun; Kumar, Chanchal; Zhang, Yanling

2007-01-01

, mitochondria, membrane, and cytosol of 3T3-L1 adipocytes. We identified 3,287 proteins while essentially eliminating false positives, making this one of the largest high confidence proteomes reported to date. Comprehensive bioinformatics analysis revealed that the adipocyte proteome, despite its specialized...

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.

Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

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Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

2018-01-01

identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

A Proteomic Approach of Bradyrhizobium/Aeschynomene Root and Stem Symbioses Reveals the Importance of the fixA Locus for Symbiosis

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Nathanael Delmotte

2014-02-01

Full Text Available Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants. Here we used a proteomics approach to investigate the bacterial physiology of photosynthetic Bradyrhizobium sp. ORS278 during the symbiotic process with the semi aquatical plant Aeschynomene indica that forms root and stem nodules. We analyzed the proteomes of bacteria extracted from each type of nodule. First, we analyzed the bacteroid proteome at two different time points and found only minor variation between the bacterial proteomes of 2-week- and 3-week-old nodules. High conservation of the bacteroid proteome was also found when comparing stem nodules and root nodules. Among the stem nodule specific proteins were those related to the phototrophic ability of Bradyrhizobium sp. ORS278. Furthermore, we compared our data with those obtained during an extensive genetic screen previously published. The symbiotic role of four candidate genes which corresponding proteins were found massively produced in the nodules but not identified during this screening was examined. Mutant analysis suggested that in addition to the EtfAB system, the fixA locus is required for symbiotic efficiency.

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

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Qun Liu

2012-01-01

Full Text Available Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale. In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

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...

A proteomics study of in vitro cyst germination and appressoria formation in ¤Phytophthora infestans¤

DEFF Research Database (Denmark)

Ebstrup, T.; Saalbach, G.; Egsgaard, H.

2005-01-01

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from ...... five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots...

Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone.

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Meier, Samuel M; Muqaku, Besnik; Ullmann, Ronald; Bileck, Andrea; Kreutz, Dominique; Mader, Johanna C; Knasmüller, Siegfried; Gerner, Christopher

2015-01-01

Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac

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.

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

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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.

Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.).

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Aranjuelo, Iker; Molero, Gemma; Erice, Gorka; Avice, Jean Christophe; Nogués, Salvador

2011-01-01

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation

Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.).

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Guo, Jinju; Wang, Peng; Cheng, Qing; Sun, Limin; Wang, Hongyu; Wang, Yutong; Kao, Lina; Li, Yanan; Qiu, Tuoyu; Yang, Wencai; Shen, Huolin

2017-09-25

Although cytoplasmic male sterility (CMS) is widely used for developing pepper hybrids, its molecular mechanism remains unclear. In this study, we used a high-throughput proteomics method called label-free to compare protein abundance across a pepper CMS line (A-line) and its isogenic maintainer line (B-line). Data are available via ProteomeXchange with identifier PXD006104. Approximately 324 differentially abundant protein species were identified and quantified; among which, 47 were up-accumulated and 140 were down-accumulated in the A-line; additionally, 75 and 62 protein species were specifically accumulated in the A-line and B-line, respectively. Protein species involved in pollen exine formation, pyruvate metabolic processes, the tricarboxylic acid cycle, the mitochondrial electron transport chain, and oxidative stress response were observed to be differentially accumulated between A-line and B-line, suggesting their potential roles in the regulation of pepper pollen abortion. Based on our data, we proposed a potential regulatory network for pepper CMS that unifies these processes. Artificial emasculation is a major obstacle in pepper hybrid breeding for its high labor cost and poor seed purity. While the use of cytoplasmic male sterility (CMS) in hybrid system is seriously frustrated because a long time is needed to cultivate male sterility line and its isogenic restore line. Transgenic technology is an effective and rapid method to obtain male sterility lines and its widely application has very important significance in speeding up breeding process in pepper. Although numerous studies have been conducted to select the genes related to male sterility, the molecular mechanism of cytoplasmic male sterility in pepper remains unknown. In this study, we used the high-throughput proteomic method called "label-free", coupled with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS), to perform a novel comparison of expression profiles in a CMS pepper line

Dynamic changes of histone H3 marks during Caenorhabditis elegans lifecycle revealed by middle-down proteomics

DEFF Research Database (Denmark)

Sidoli, Simone; Vandamme, Julien; Elisabetta Salcini, Anna

2016-01-01

We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval......-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during...... that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms. This article is protected by copyright. All...

Mining the active proteome of Arabidopsis thaliana

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Renier A. L. Van Der Hoorn

2011-11-01

Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

IgV peptide mapping of native Ro60 autoantibody proteomes in primary Sjögren's syndrome reveals molecular markers of Ro/La diversification.

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

2016-12-01

We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity. Copyright © 2016 Elsevier Inc. All rights reserved.

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...

Global Proteome Analysis of the NCI-60 Cell Line Panel

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Amin Moghaddas Gholami

2013-08-01

Full Text Available The NCI-60 cell line collection is a very widely used panel for the study of cellular mechanisms of cancer in general and in vitro drug action in particular. It is a model system for the tissue types and genetic diversity of human cancers and has been extensively molecularly characterized. Here, we present a quantitative proteome and kinome profile of the NCI-60 panel covering, in total, 10,350 proteins (including 375 protein kinases and including a core cancer proteome of 5,578 proteins that were consistently quantified across all tissue types. Bioinformatic analysis revealed strong cell line clusters according to tissue type and disclosed hundreds of differentially regulated proteins representing potential biomarkers for numerous tumor properties. Integration with public transcriptome data showed considerable similarity between mRNA and protein expression. Modeling of proteome and drug-response profiles for 108 FDA-approved drugs identified known and potential protein markers for drug sensitivity and resistance. To enable community access to this unique resource, we incorporated it into a public database for comparative and integrative analysis (http://wzw.tum.de/proteomics/nci60.

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

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Gao, Yanpan; Chen, Yanyu; Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

2017-01-31

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections.

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Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei

2017-07-01

Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

2017-04-01

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

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

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Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

2015-10-30

Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

International Nuclear Information System (INIS)

Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

2015-01-01

Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

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Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan

2015-08-03

Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

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Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

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Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

2014-11-01

Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

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Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

2013-03-01

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

A proteomics study reveals a predominant change in MaoB expression in platelets of healthy volunteers after high protein meat diet

DEFF Research Database (Denmark)

Zellner, Maria; Babeluk, Rita; Jakobsen, Lene Holm

2011-01-01

Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP...... reproducibly studied platelet proteins only the level of monoamine oxidase B (MaoB), a neurotransmitter degrading enzyme, decreased by 26% significantly (adjusted P value diet. In addition, we found a correlation (r = 0.477; P ...) diet on both cognitive performance and corresponding biochemical parameters. A randomised intervention study was conducted with 23 healthy males (aged 19-31 years) to investigate the effects of a usual (UP) versus a HP diet on cognitive function and on the platelet proteome a well-established model...

Proteomic study related to vascular connections in watermelon scions grafted onto bottle-gourd rootstock under different light intensities.

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Sowbiya Muneer

Full Text Available Although grafting is broadly used in the production of crops, no information is available about the proteins involved in vascular connections between rootstock and scion. Similarly, proteome changes under the light intensities widely used for grafted seedlings are of practical use. The objective of this study was to determine the proteome of vascular connections using watermelon (Citrullus vulgaris Schrad. 'Sambok Honey' and 'Speed' as the scion and bottle gourd (Lagenaria siceraria Stanld. 'RS Dongjanggun' as the rootstock grown under different light intensities (25, 50, 75 and 100 μmol m-2 s-1. Our proteomic analysis revealed 24 and 27 differentially expressed proteins in 'Sambok Honey' and 'Speed', respectively, under different light intensities. The identified proteins were largely involved in ion binding, amino acid metabolism, transcriptional regulation and defense response. The enhancement of ion-binding, transcriptional regulation, amino acid metabolism, and defense response proteins suggests a strengthening of the connection between the rootstock and scion under high light intensity. Indeed, the accumulation of key enzymes in the biological processes described above appears to play an important role in the vascular connections of grafted seedlings. Moreover, it appears that 100 μmol m-2 s-1 results in better protein expression responses in grafted seedlings.

Study on the effect of irradiation on algae by proteomics

International Nuclear Information System (INIS)

Choi, Jong Il; Yoon, Yo Han; Kim, Jae Hun

2010-06-01

Algae has been utilized as food material from long time ago, and recently newly recognized as functional materials and the source of bio-fuel. But, the study on the algae is just beginning and the study on protein expression and growth by the change of condition was not reported. In this study, the effect of radiation on the protein expression was investigated for the protection mechanisms and new genome source and furthermore, isolation of new mutant strains. To monitor the growth of algae, absorbance and FDA staining methods were developed and the content of lipid of algae species were measured. With these methods, the radiation sensitivity of algae species was determined. To investigate the proteome of algae, 2D-electrophoresis methods was applied. From the comparison of proteomes, the radiation specific expressed protein was identified as thioredoxin-h and its nucleotide sequences was defined. The expression of thioredoxin-h was further defined on the mRNA level. Also, the extract of algae species was analyzed for its antioxidant activity and polyphenolic content. The changes in antioxidant activity of extract by radiation was investigated. From the radiation experiments, mutant Spirogyra species having higher resistant against radical stress was obtained. The mutant strain has higher antioxidant activity. This results can provide the proteome date and mutation technology of algae and further contribute in the activation of fishery industry and national health enhancement

Proteomic study on gender differences in aging kidney of mice

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Cristobal Susana

2009-04-01

Full Text Available Abstract Background This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS. Results This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes. Conclusion Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.

Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

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Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

2015-01-01

Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

Environmental proteomics reveals early microbial community responses to biostimulation at a uranium- and nitrate-contaminated site

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Chourey, Karuna [ORNL; Nissen, Silke [ORNL; Vishnivetskaya, T. [University of Tennessee, Knoxville (UTK); Shah, Manesh B [ORNL; Pffifner, Susan [University of Tennessee, Knoxville (UTK); Hettich, Robert {Bob} L [ORNL; Loeffler, Frank E [ORNL

2013-01-01

High performance mass spectrometry instrumentation coupled with improved protein extraction techniques enable metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e., 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e., Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences in protein expression between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected up-gradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate (PHB) granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of a Dechloromonas sp. was highly expressed suggesting active nitrous oxide (N2O) respiration. c-type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that metal reduction has not commenced 4 days post EVO delivery. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique for complementing nucleic acid-based approaches.

The proteomic complexity and rise of the primordial ancestor of diversified life

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Kim Kyung

2011-05-01

Full Text Available Abstract Background The last universal common ancestor represents the primordial cellular organism from which diversified life was derived. This urancestor accumulated genetic information before the rise of organismal lineages and is considered to be either a simple 'progenote' organism with a rudimentary translational apparatus or a more complex 'cenancestor' with almost all essential biological processes. Recent comparative genomic studies support the latter model and propose that the urancestor was similar to modern organisms in terms of gene content. However, most of these studies were based on molecular sequences, which are fast evolving and of limited value for deep evolutionary explorations. Results Here we engage in a phylogenomic study of protein domain structure in the proteomes of 420 free-living fully sequenced organisms. Domains were defined at the highly conserved fold superfamily (FSF level of structural classification and an iterative phylogenomic approach was used to reconstruct max_set and min_set FSF repertoires as upper and lower bounds of the urancestral proteome. While the functional make up of the urancestral sets was complex, they represent only 5-11% of the 1,420 FSFs of extant proteomes and their make up and reuse was at least 5 and 3 times smaller than proteomes of free-living organisms, repectively. Trees of proteomes reconstructed directly from FSFs or from molecular functions, which included the max_set and min_set as articial taxa, showed that urancestors were always placed at their base and rooted the tree of life in Archaea. Finally, a molecular clock of FSFs suggests the min_set reflects urancestral genetic make up more reliably and confirms diversified life emerged about 2.9 billion years ago during the start of planet oxygenation. Conclusions The minimum urancestral FSF set reveals the urancestor had advanced metabolic capabilities, was especially rich in nucleotide metabolism enzymes, had pathways for the

Proteome reference map of Drosophila melanogaster head.

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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.

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.

Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

Science.gov (United States)

Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

2017-06-01

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

Science.gov (United States)

Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

2013-01-01

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

ProteomicsDB.

Science.gov (United States)

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.

A Pilot Proteomic Analysis of Salivary Biomarkers in Autism Spectrum Disorder.

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Ngounou Wetie, Armand G; Wormwood, Kelly L; Russell, Stefanie; Ryan, Jeanne P; Darie, Costel C; Woods, Alisa G

2015-06-01

Autism spectrum disorder (ASD) prevalence is increasing, with current estimates at 1/68-1/50 individuals diagnosed with an ASD. Diagnosis is based on behavioral assessments. Early diagnosis and intervention is known to greatly improve functional outcomes in people with ASD. Diagnosis, treatment monitoring and prognosis of ASD symptoms could be facilitated with biomarkers to complement behavioral assessments. Mass spectrometry (MS) based proteomics may help reveal biomarkers for ASD. In this pilot study, we have analyzed the salivary proteome in individuals with ASD compared to neurotypical control subjects, using MS-based proteomics. Our goal is to optimize methods for salivary proteomic biomarker discovery and to identify initial putative biomarkers in people with ASDs. The salivary proteome is virtually unstudied in ASD, and saliva could provide an easily accessible biomaterial for analysis. Using nano liquid chromatography-tandem mass spectrometry, we found statistically significant differences in several salivary proteins, including elevated prolactin-inducible protein, lactotransferrin, Ig kappa chain C region, Ig gamma-1 chain C region, Ig lambda-2 chain C regions, neutrophil elastase, polymeric immunoglobulin receptor and deleted in malignant brain tumors 1. Our results indicate that this is an effective method for identification of salivary protein biomarkers, support the concept that immune system and gastrointestinal disturbances may be present in individuals with ASDs and point toward the need for larger studies in behaviorally-characterized individuals. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

Proteomic analysis revealed alterations of the Plasmodium falciparum metabolism following salicylhydroxamic acid exposure

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Torrentino-Madamet M

2011-09-01

Full Text Available Marylin Torrentino-Madamet1, Lionel Almeras2, Christelle Travaillé1, Véronique Sinou1, Matthieu Pophillat3, Maya Belghazi4, Patrick Fourquet3, Yves Jammes5, Daniel Parzy11UMR-MD3, Université de la Méditerranée, Antenne IRBA de Marseille (IMTSSA, Le Pharo, 2Unité de Recherche en Biologie et Epidémiologie Parasitaires, Antenne IRBA de Marseille (IMTSSA, Le Pharo, 3Centre d'Immunologie de Marseille Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, 4Centre d'Analyse Protéomique de Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, 5UMR-MD2, Physiologie et Physiopathologie en Conditions d'Oxygénations Extrêmes, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, Marseille, FranceObjectives: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a “branched respiratory chain.” This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM, showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure.Design: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed.Results: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins

Proteomic analysis reveals age-related changes in tendon matrix composition, with age- and injury-specific matrix fragmentation.

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Peffers, Mandy J; Thorpe, Chavaunne T; Collins, John A; Eong, Robin; Wei, Timothy K J; Screen, Hazel R C; Clegg, Peter D

2014-09-12

Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Proteomic-Based Approaches for the Study of Cytokines in Lung Cancer

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Ángela Marrugal

2016-01-01

Full Text Available Proteomic techniques are currently used to understand the biology of different human diseases, including studies of the cell signaling pathways implicated in cancer progression, which is important in knowing the roles of different proteins in tumor development. Due to its poor prognosis, proteomic approaches are focused on the identification of new biomarkers for the early diagnosis, prognosis, and targeted treatment of lung cancer. Cytokines are proteins involved in inflammatory processes and have been proposed as lung cancer biomarkers and therapeutic targets because it has been reported that some cytokines play important roles in tumor development, invasion, and metastasis. In this review, we aim to summarize the different proteomic techniques used to discover new lung cancer biomarkers and therapeutic targets. Several cytokines have been identified as important players in lung cancer using these techniques. We underline the most important cytokines that are useful as biomarkers and therapeutic targets. We also summarize some of the therapeutic strategies targeted for these cytokines in lung cancer.

The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide

DEFF Research Database (Denmark)

Cassoli, Juliana Silva; Verano-Braga, Thiago; Oliveira, Joacir Stolarz

2013-01-01

duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low...... present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy...

Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

OpenAIRE

Wang, Yong-Qiang; Yang, Yong; Fei, Zhangjun; Yuan, Hui; Fish, Tara; Thannhauser, Theodore W.; Mazourek, Michael; Kochian, Leon V.; Wang, Xiaowu; Li, Li

2013-01-01

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953?2262 proteins from chromoplasts of diff...

Soybean Proteome Database 2012: Update on the comprehensive data repository for soybean proteomics

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Hajime eOhyanagi

2012-05-01

Full Text Available The Soybean Proteome Database (SPD was created to provide a data repository for functional analyses of soybean responses to flooding stress, thought to be a major constraint for establishment and production of this plant. Since the last publication of the SPD, we thoroughly enhanced the contents of database, particularly protein samples and their annotations from several organelles. The current release contains 23 reference maps of soybean (Glycine max cv. Enrei proteins collected from several organs, tissues and organelles including the maps for plasma membrane, cell wall, chloroplast and mitochondrion, which were electrophoresed on two-dimensional polyacrylamide gels. Furthermore, the proteins analyzed with gel-free proteomics technique have been added and available online. In addition to protein fluctuations under flooding, those of salt and drought stress have been included in the current release. An omics table also has been provided to reveal relationships among mRNAs, proteins and metabolites with a unified temporal-profile tag in order to facilitate retrieval of the data based on the temporal profiles. An intuitive user interface based on dynamic HTML enables users to browse the network as well as the profiles of multiple omes in an integrated fashion. The SPD is available at: http://proteome.dc.affrc.go.jp/Soybean/.

Comparative proteomic and metabolomic analysis reveal the antiosteoporotic molecular mechanism of icariin from Epimedium brevicornu maxim.

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Xue, Liming; Jiang, Yiping; Han, Ting; Zhang, Naidan; Qin, Luping; Xin, Hailiang; Zhang, Qiaoyan

2016-11-04

Icariin, a principal flavonoid glycoside of Epimedium brevicornu Maxim, has been widely proved to possess antiosteoporotic activity with promoting bone formation and decreasing bone resorption. However, the involving mechanisms remain unclear. To clear a global insight of signal pathways involved in anti-osteoporotic mechanism of icariin at proteins and metabolites level by integrating the proteomics and NMR metabonomics, in a systems biology approach. Mice were divided into sham, OVX model and icariin-treated OVX group, after 90 days treatment, difference gel electrophoresis combined with MALDI-TOF/TOF proteomics analysis on bone femur and serum metabolomics were carried out for monitor intracellular processes and elucidate anti-osteoporotic mechanism of icariin. Osteoblast and osteoclast were applied to evaluate the potential signal pathways. Twenty three proteins in bone femur, and 8 metabolites in serum, were significantly altered and identified, involving in bone remodeling, energy metabolism, cytoskeleton, lipid metabolism, MAPK signaling, Ca 2+ signaling et, al. Furthermore, animal experiment show icariin could enhance the BMD and BMC, decrease CTX-I level in ovariectomized mice. The mitochondrial membrane potential and the intracellular ATP levels were increased significantly, and the cytoskeleton were improved in icariin-treatment osteoblast and osteoclast. Icariin also increased mRNA expression of Runx2 and osterix of OB, decreased CTR and CAII mRNA expression and protein expression of P38 and JNK. However, icariin did not reveal any inhibition of the collagenolytic activity of cathepsin K, mRNA expression of MMP-9 and protein expression of ERK in osteoclast. we consider icariin as multi-targeting compounds for treating with osteoporosis, involve initiating osteoblastogenesis, inhibiting adipogenesis, and preventing osteoclast differentiation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features

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Carvalho Carla M

2012-01-01

Full Text Available Abstract Background Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies. Methods vB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry. Results and conclusions The DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and ϕ29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle. Conclusions Genes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS degradation, which has not been reported

Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

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Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

2013-01-22

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

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

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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

Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings*

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Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A.; Chen, Wei; Yang, Yong; Rose, Jocelyn K. C.; Zhang, Sheng; Yi, Gan-Jun

2012-01-01

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

Science.gov (United States)

Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A; Chen, Wei; Yang, Yong; Rose, Jocelyn K C; Zhang, Sheng; Yi, Gan-Jun

2012-12-01

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

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Marangoni Sérgio

2009-04-01

Full Text Available Abstract Background Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area – BA22p identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6 and glial fibrillary acidic protein (GFAP were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Radiation induced changes in proteome of mice jejunum: an in vivo 2DE study

International Nuclear Information System (INIS)

Bajaj, Sania; Dutta, Ajaswrata; Gupta, Manju L.

2014-01-01

Radiation exposure results in severe damage to biological system, by affecting cellular macromolecules of an individual. Damage to bio-molecules may lead to up/down-regulation of proteins, leading to dysfunction of organs. Gastrointestinal tract a key organ for digestion, absorption and barrier to the luminal bacteria and toxins, is one of the highly sensitive radiosensitive organ. Current study is focused on differential proteomic approach to understand the effect of radiation on intestinal (jejunum) proteins in a time dependent manner. Experiments were carried out initially to determine the appropriate conditions for separation of proteins in GI tissue of non irradiated control male C57BL6/J mice. 8-10 weeks old animals were exposed to 9 Gy (lethal) dose of gamma radiation. Differential expression of gastrointestinal tissue (jejunum) proteome was studied by 2DE at different time intervals. The intensity of protein spots of different treatment groups and control was measured by PD Quest software and the differential expression of respective proteins was calculated manually. Comparison of 2-DE gel images of irradiated jejunum tissue samples showed differential expression of various proteins when compared with untreated samples. A significant upregulation of total protein spots was observed within 1 hr group of 9 Gy radiation exposed sample and maximum down-regulation was evident at 72 hr. Out of 24 spots identified in the irradiated samples, 15 spots were down-regulated, and 3 spots were found missing in 72 hr group of irradiated samples respectively. Time dependent regulation of protein expression in irradiated jejunum was thus prominently evident. The data obtained from the present study has revealed differential radio sensitivity of some of the proteins which certainly have a definite role in inducing major cellular changes after radiation exposure. The finding also suggests that proteomic approach could be a potential tool to access the role of specific

Quantitative proteome profiling of human myoma and myometrium tissue reveals kinase expression signatures with potential for therapeutic intervention

NARCIS (Netherlands)

Lemeer, Simone; Gholami, Amin Moghaddas; Wu, Zhixiang; Kuster, Bernhard

2015-01-01

Uterine leiomyomas are benign tumors affecting a large proportion of the female population. Despite the very high prevalence, the molecular basis for understanding the onset and development of the disease are still poorly understood. In this study, we profiled the proteomes and kinomes of leiomyoma

A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles

DEFF Research Database (Denmark)

Wagner, Sebastian Alexander; Beli, Petra; Weinert, Brian Tate

2011-01-01

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single...

Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

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

2015-05-01

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

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

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

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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.

Toxic effects of male Perna viridis gonad exposed to BaP, DDT and their mixture: A metabolomic and proteomic study of the underlying mechanism.

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Song, Qinqin; Zheng, Pengfei; Qiu, Liguo; Jiang, Xiu; Zhao, Hongwei; Zhou, Hailong; Han, Qian; Diao, Xiaoping

2016-01-05

Benzo(a)pyrene and dichlorodiphenyltrichloroethane are typical persistent organic pollutants, and also the widespread environmental estrogens with known toxicity towards green mussels Perna viridis. In this study, the toxicological effects of BaP and DDT and their mixture were assessed in green mussel gonads using proteomic and metabolomic approaches. Metabolomics by NMR spectroscopy revealed that BaP did not show obvious metabolite changes in the gonad of male green mussel. DDT mainly caused some disturbance of osmotic regulation and energy metabolism by changing BCAAs, alanine, threonine, arginine, etc., unknown metabolite (3.53 ppm), glycine, homarine and ATP at different levels. However, the mixture of BaP and DDT mainly caused some disturbance in osmotic regulation and energy metabolism by differentially altering branched chain amino acids, glutamate, alanine, arginine, unknown metabolite (3.53 ppm), glycine, 4-aminobutyrate, dimethylglycine, homarine and ATP. The results suggest that DDT alone may cause most of metabolites changes in the mixture exposed male mussel gonad, and the results also show that the male P. viridis gonad was more sensitive to DDT than BaP exposures. Proteomic study showed that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced signal transduction, oxidative stress, spermatogenesis, etc. in the male green mussel gonad; whereas DDT exposure altered proteins that were associated with signal transduction, oxidative stress, cytoskeleton and cell structure, cellular organization, energy metabolism, etc. However, the mixture of BaP and DDT affected proteins related to cytoskeleton and cell structure, oxidative stress, cellular organization, etc. This research demonstrated that metabolomic and proteomic approaches could better elucidate the underlying mechanism of environmental pollutants gonad toxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Neutrophil proteomic analysis reveals the participation of antioxidant enzymes, motility and ribosomal proteins in the prevention of ischemic effects by preconditioning

DEFF Research Database (Denmark)

Arshid, S; Tahir, M; Fontes, B

2017-01-01

therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome...... in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding...

Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism

DEFF Research Database (Denmark)

Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill

2015-01-01

Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular...

Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants.

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Yu, Wencheng; Chen, Zhen; Shen, Liang; Wang, Yuanpeng; Li, Qingbiao; Yan, Shan; Zhong, Chuan-Jian; He, Ning

2016-04-01

Some bioflocculants composed of extracellular polymeric substances are produced under peculiar conditions. Bacillus licheniformis CGMCC2876 is a microorganism that secretes both extracellular polysaccharides (EPS) and poly-gamma-glutamic acid (γ-PGA) under stress conditions. In this work, SWATH acquisition LC-MS/MS method was adopted for differential proteomic analysis of B. licheniformis, aiming at determining the bacterial stress mechanism. Compared with LB culture, 190 differentially expressed proteins were identified in B. licheniformis CGMCC2876 cultivated in EPS culture, including 117 up-regulated and 73 down-regulated proteins. In γ-PGA culture, 151 differentially expressed proteins, 89 up-regulated and 62 down-regulated, were found in the cells. Up-regulated proteins involved in amino acid biosynthesis were found to account for 43% and 41% of the proteomes in EPS and γ-PGA cultivated cells, respectively. Additionally, a series of proteins associated with amino acid degradation were found to be repressed under EPS and γ-PGA culture conditions. Transcriptional profiling via the qPCR detection of selected genes verified the proteomic analysis. Analysis of free amino acids in the bacterial cells further suggested the presence of amino acid starvation conditions. EPS or γ-PGA was synthesized to alleviate the effect of amino acid limitation in B. licheniformis. This study identified a stress response mechanism in the synthesis of macromolecules in B. licheniformis, providing potential culture strategies to improve the production of two promising bioflocculants. © 2015 Wiley Periodicals, Inc.

Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

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Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman

2012-02-02

Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. Copyright © 2011 Elsevier B.V. All rights reserved.

Detergents: Friends not foes for high-performance membrane proteomics toward precision medicine.

Science.gov (United States)

Zhang, Xi

2017-02-01

Precision medicine, particularly therapeutics, emphasizes the atomic-precise, dynamic, and systems visualization of human membrane proteins and their endogenous modifiers. For years, bottom-up proteomics has grappled with removing and avoiding detergents, yet faltered at the therapeutic-pivotal membrane proteins, which have been tackled by classical approaches and are known for decades refractory to single-phase aqueous or organic denaturants. Hydrophobicity and aggregation commonly challenge tissue and cell lysates, biofluids, and enriched samples. Frequently, expected membrane proteins and peptides are not identified by shotgun bottom-up proteomics, let alone robust quantitation. This review argues the cause of this proteomic crisis is not detergents per se, but the choice of detergents. Recently, inclusion of compatible detergents for membrane protein extraction and digestion has revealed stark improvements in both quantitative and structural proteomics. This review analyzes detergent properties behind recent proteomic advances, and proposes that rational use of detergents may reconcile outstanding membrane proteomics dilemmas, enabling ultradeep coverage and minimal artifacts for robust protein and endogenous PTM measurements. The simplicity of detergent tools confers bottom-up membrane proteomics the sophistication toward precision medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lessons from the proteomic study of osteoarthritis.

Science.gov (United States)

Ruiz-Romero, Cristina; Fernández-Puente, Patricia; Calamia, Valentina; Blanco, Francisco J

2015-08-01

Osteoarthritis is the most common rheumatic pathology and one of the leading causes of disability worldwide. It is a very complex disease whose etiopathogenesis is not fully understood. Furthermore, there are serious limitations for its management, since it lacks specific and sensitive biomarkers for early diagnosis, prognosis and therapeutic monitoring. Proteomic approaches performed in the last few decades have contributed to the knowledge on the molecular mechanisms that participate in this pathology and they have also led to interesting panels of putative biomarker candidates. In the next few years, further efforts should be made for translating these findings into the clinical routines. It is expected that targeted proteomics strategies will be highly valuable for the verification and qualification of biomarkers of osteoarthritis.

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...

Proteome analysis of ofloxacin and moxifloxacin induced mycobacterium tuberculosis isolates by proteomic approach.

Science.gov (United States)

Lata, Manju; Sharma, Divakar; Kumar, Bhavnesh; Deo, Nirmala; Tiwari, Pramod Kumar; Bisht, Deepa; Venkatesan, Krishnamurthy

2015-01-01

Ofloxacin (OFX) and moxifloxacin (MOX) are the most promising second line drugs for tuberculosis treatment. Although the primary mechanism of action of OFX and MOX is gyrase inhibition, other possible mechanisms cannot be ruled out. Being the functional moiety of cell, the proteins act as primary targets for developing drugs, diagnostics and therapeutics. In this study we have investigated the proteomic changes of Mycobacterium tuberculosis isolates induced by OFX and MOX by applying comparative proteomic approaches based on two-dinensional gel electrophoresis (2DE) along with matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF/TOF-MS) and bioinformatic tools. The findings are likely to provide new understanding of OFX and MOX mechanisms that might be helpful in exploring new diagnostics and drug targets. Our study explored eleven proteins (Rv2889c, Rv2623, Rv0952, Rv1827, Rv1932, Rv0054, Rv1080c, Rv3418c, Rv3914, Rv1636 and Rv0009) that were overexpressed in the presence of drugs. Among them, Rv2623, Rv1827 and Rv1636 were identified as proteins with unknown function. InterProScan and molecular docking revealed that the conserved domain of hypothetical proteins interact with OFX and MOX which indicate a probable inhibition/modulation of the functioning of these proteins by both drugs, which might be overexpressed to overcome this effect.

Proteomic analysis of Sydney Rock oysters (Saccostrea glomerata) exposed to metal contamination in the field

International Nuclear Information System (INIS)

Thompson, Emma L.; Taylor, Daisy A.; Nair, Sham V.; Birch, Gavin; Hose, Grant C.; Raftos, David A.

2012-01-01

This study used proteomics to assess the impacts of metal contamination in the field on Sydney Rock oysters. Oysters were transplanted into Lake Macquarie, NSW, for two weeks in both 2009 and 2010. Two-dimensional electrophoresis identified changes in protein expression profiles of oyster haemolymph between control and metal contaminated sites. There were unique protein expression profiles for each field trial. Principal components analysis attributed these differences in oyster proteomes to the different combinations and concentrations of metals and other environmental variables present during the three field trials. Identification of differentially expressed proteins showed that proteins associated with cytoskeletal activity and stress responses were the most commonly affected biological functions in the Sydney Rock oyster. Overall, the data show that proteomics combined with multivariate analysis has the potential to link the effects of contaminants with biological consequences. - Highlights: ► Sydney Rock oyster haemolymph was analysed by proteomics after metal exposure in 3 field trials. ► 2-DE analysis was used to compare protein profiles between control and contaminated sites. ► Different protein expression profiles were revealed per field trial. ► Principal components analysis attributed profiles to different suites of metals and environmental variables per trial. ► The study highlights the need to do multiple field trials and to combine proteomic and enviro. data. - This study used proteomics to analyse impacts of metal contamination on Sydney Rock oyster (Saccostrea glomerata) haemolymph in multiple field trials.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

Science.gov (United States)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

Science.gov (United States)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-01-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery. PMID:26956660

Proteomic Signatures of Thymomas.

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Linan Wang

Full Text Available Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas.

Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics

NARCIS (Netherlands)

Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic Y; Jennings, Paul

2015-01-01

Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to

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....

A comparative proteomic study on the effects of metal pollution in oysters Crassostrea hongkongensis.

Science.gov (United States)

Xu, Lanlan; Ji, Chenglong; Wu, Huifeng; Tan, Qiaoguo; Wang, Wen-Xiong

2016-11-15

The metal pollution has posed great risk on the coastal organisms along the Jiulongjiang Estuary in South China. In this work, two-dimensional electrophoresis-based proteomics was applied to the oysters Crassostrea hongkongensis from metal pollution sites to characterize the proteomic responses to metal pollution. Metal accumulation and proteomic responses indicated that the oysters from BJ site were more severely contaminated than those from FG site. Compared with those oyster samples from the clean site (JZ), metal pollution induced cellular injuries, oxidative and immune stresses in oyster heapatopancreas from both BJ and FG sites via differential metabolic pathways. In addition, metal pollution in BJ site induced disturbance in energy and lipid metabolisms in oysters. Results indicated that cathepsin L and ferritin GF1 might be the biomarkers of As and Fe in oyster C. hongkongensis, respectively. This study demonstrates that proteomics is a useful tool for investigating biological effects induced by metal pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

Science.gov (United States)

Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.

Science.gov (United States)

Woo, Patrick C Y; Lau, Susanna K P; Tse, Herman; Teng, Jade L L; Curreem, Shirly O T; Tsang, Alan K L; Fan, Rachel Y Y; Wong, Gilman K M; Huang, Yi; Loman, Nicholas J; Snyder, Lori A S; Cai, James J; Huang, Jian-Dong; Mak, William; Pallen, Mark J; Lok, Si; Yuen, Kwok-Yung

2009-03-01

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors-such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases-are present. Proteomes of L. hongkongensis HLHK9 cultured at 37 degrees C (human body temperature) and 20 degrees C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)-NAGK-20 and NAGK-37-in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20 degrees C, whereas NAGK-37 showed higher expression at 37 degrees C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other

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

Science.gov (United States)

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.

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...

PROTEOMIC PROFILE REVEALS THE DIVERSITY AND COMPLEXITY OF LEAF PROTEINS IN SPINACH (BETA VULGARIS VAR. ALL GREEN

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Sudip Ghosh

2016-06-01

Full Text Available Leaf is a source organ that serves dual function in photosynthesis and transpiration. As a primary interface between plant and ecosystem, it performs a range of biological processes from carbon assimilation and metabolite partitioning to plant productivity. Basic features of the leaf functionality are conserved in angiosperms exhibiting common and unique characteristics. Spinach has been the model crop for studying leaf function, primarily photosynthesis. It is a reservoir of several hundreds of primary and secondary biomolecules. To better understand the molecular basis for photochemical reaction and metabolic partitioning, we developed leaf proteome of Indian spinach (Beta vulgaris var. all green. LC-ESI-MS/MS analysis identified 639 proteins exhibiting discrete molecular features and functions, including photosynthesis, transpiration, gaseous exchange, transport, redox status, cell defense, and floral induction besides the presence of proteins with unknown function. This represents the first comprehensive foliage proteome of green leafy vegetable. Together, this work provides important insights into the molecular networks underlying spinach leaf biological processes.

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

Science.gov (United States)

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

2013-01-14

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

Studying Different Clinical Syndromes Of Paediatric Severe Malaria Using Plasma Proteomics

KAUST Repository

Ramaprasad, Abhinay

2012-08-01

Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

Individual variability in the venom proteome of juvenile Bothrops jararaca specimens.

Science.gov (United States)

Dias, Gabriela S; Kitano, Eduardo S; Pagotto, Ana H; Sant'anna, Sávio S; Rocha, Marisa M T; Zelanis, André; Serrano, Solange M T

2013-10-04

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Science.gov (United States)

Quecine, Maria Carolina; Leite, Thiago Falda; Bini, Andressa Peres; Regiani, Thais; Franceschini, Lívia Maria; Budzinski, Ilara Gabriela Frasson; Marques, Felipe Garbelini; Labate, Mônica Teresa Veneziano; Guidetti-Gonzalez, Simone; Moon, David Henry; Labate, Carlos Alberto

2016-01-01

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Directory of Open Access Journals (Sweden)

Maria Carolina Quecine

Full Text Available Puccinia psidii sensu lato (s.l. is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava and eucalyptus leaves (PpEucalyptus. NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

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.

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.

Temporal proteomic analysis reveals defects in small-intestinal development of porcine fetuses with intrauterine growth restriction.

Science.gov (United States)

Wang, Xiaoqiu; Lin, Gang; Liu, Chuang; Feng, Cuiping; Zhou, Huaijun; Wang, Taiji; Li, Defa; Wu, Guoyao; Wang, Junjun

2014-07-01

The fetus/neonate with intrauterine growth restriction (IUGR) has a high perinatal mortality and morbidity rate, as well as reduced efficiency for nutrients utilization. Our previous studies showed alterations of intestinal proteome in IUGR piglets both at birth and during the nursing period. Considering the potential long-term impacts of fetal programming and substantial increases in amounts of amniotic fluid nutrients from mid-gestation in pigs, the present study involved IUGR porcine fetuses from days 60 to 110 of gestation (mid to late gestation). We identified 59 differentially expressed proteins in the fetal small intestine that are related to intestinal growth, development and reprogramming. Our results further indicated increased abundances of proteins and enzymes associated with oxidative stress, apoptosis and protein degradation, as well as decreased abundances of proteins that are required for maintenance of cell structure and motility, absorption and transport of nutrients, energy metabolism, and protein synthesis in the fetal gut. Moreover, IUGR from middle to late gestation was associated with reduced expression of intestinal proteins that participate in regulation of gene expression and signal transduction. Collectively, these findings provide the first evidence for altered proteomes in the small intestine of IUGR fetuses, thereby predisposing the gut to metabolic defects during gestation and neonatal periods. Copyright © 2014 Elsevier Inc. All rights reserved.

Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics

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Sijia Wei

2017-05-01

Full Text Available Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway, nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein–protein interaction (PPI networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

Science.gov (United States)

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

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

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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.

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.

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.

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.

Recent mass spectrometry-based proteomics for biomarker discovery in lung cancer, COPD, and asthma.

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Fujii, Kiyonaga; Nakamura, Haruhiko; Nishimura, Toshihide

2017-04-01

Lung cancer and related diseases have been one of the most common causes of deaths worldwide. Genomic-based biomarkers may hardly reflect the underlying dynamic molecular mechanism of functional protein interactions, which is the center of a disease. Recent developments in mass spectrometry (MS) have made it possible to analyze disease-relevant proteins expressed in clinical specimens by proteomic challenges. Areas covered: To understand the molecular mechanisms of lung cancer and its subtypes, chronic obstructive pulmonary disease (COPD), asthma and others, great efforts have been taken to identify numerous relevant proteins by MS-based clinical proteomic approaches. Since lung cancer is a multifactorial disease that is biologically associated with asthma and COPD among various lung diseases, this study focused on proteomic studies on biomarker discovery using various clinical specimens for lung cancer, COPD, and asthma. Expert commentary: MS-based exploratory proteomics utilizing clinical specimens, which can incorporate both experimental and bioinformatic analysis of protein-protein interaction and also can adopt proteogenomic approaches, makes it possible to reveal molecular networks that are relevant to a disease subgroup and that could differentiate between drug responders and non-responders, good and poor prognoses, drug resistance, and so on.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

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El-Bebany, Ahmed F; Rampitsch, Christof; Daayf, Fouad

2010-01-01

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle.

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Horcajo, P; Xia, D; Randle, N; Collantes-Fernández, E; Wastling, J; Ortega-Mora, L M; Regidor-Cerrillo, J

2017-11-14

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study

DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration.

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Sina Haas

Full Text Available Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

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Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

2015-10-30

Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

A DIGE proteomic analysis for high-intensity exercise-trained rat skeletal muscle.

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Yamaguchi, Wataru; Fujimoto, Eri; Higuchi, Mitsuru; Tabata, Izumi

2010-09-01

Exercise training induces various adaptations in skeletal muscles. However, the mechanisms remain unclear. In this study, we conducted 2D-DIGE proteomic analysis, which has not yet been used for elucidating adaptations of skeletal muscle after high-intensity exercise training (HIT). For 5 days, rats performed HIT, which consisted of 14 20-s swimming exercise bouts carrying a weight (14% of the body weight), and 10-s pause between bouts. The 2D-DIGE analysis was conducted on epitrochlearis muscles excised 18 h after the final training exercise. Proteomic profiling revealed that out of 800 detected and matched spots, 13 proteins exhibited changed expression by HIT compared with sedentary rats. All proteins were identified by MALDI-TOF/MS. Furthermore, using western immunoblot analyses, significantly changed expressions of NDUFS1 and parvalbumin (PV) were validated in relation to HIT. In conclusion, the proteomic 2D-DIGE analysis following HIT-identified expressions of NDUFS1 and PV, previously unknown to have functions related to exercise-training adaptations.

Studying Different Clinical Syndromes Of Paediatric Severe Malaria Using Plasma Proteomics

KAUST Repository

Ramaprasad, Abhinay

2012-01-01

challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

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Shin, Kwang-Soo, E-mail: shinks@dju.kr [Division of Life Science, Daejeon University, Daejeon, 300-716 (Korea, Republic of); Kim, Young Hwan [Biomedical Omics Team, Korea Basic Science Institute (KBSI), Ohcang, 368-883 (Korea, Republic of); Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 305-333 (Korea, Republic of); Yu, Jae-Hyuk, E-mail: jyu1@wisc.edu [Departments of Bacteriology and Genetics, The University of Wisconsin–Madison, Madison, WI, 53706 (United States)

2015-07-31

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

International Nuclear Information System (INIS)

Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

2015-01-01

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

Science.gov (United States)

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

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.

Predicted sub-populations in a marine shrimp proteome as revealed by combined EST and cDNA data from multiple Penaeus species

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Kotewong Rattanawadee

2010-11-01

Full Text Available Abstract Background Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus vannamei, Penaeus monodon, Penaeus (Fenneropenaeus chinensis, and Penaeus (Marsupenaeus japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda, the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used. Findings Similarity searches from EST collections of the 4 shrimp species matched 64% of the protein sequences of the fruit fly, but only 45% of nematode proteins, indicating that the shrimp proteome content is more similar to that of an insect than a nematode. Combined results with 4 additional non-shrimp crustaceans increased matching to 78% of fruit fly and 56% of nematode proteins, suggesting that present shrimp EST collections still lack sequences for many conserved crustacean proteins. Analysis of matching data revealed the presence of 4 EST groups from shrimp, namely sequences for proteins that are both fruit fly-like and nematode-like, fruit fly-like only, nematode-like only, and non-matching. Gene ontology profiles of proteins for the 3 matching EST groups were analyzed. For non-matching ESTs, a small fraction matched protein sequences from other species in the UniProt database, including other crustacean-specific proteins. Conclusions Shrimp ESTs indicated that the shrimp proteome is comprised of sub-populations of proteins similar to those common to both insect and nematode models, those present specifically in either model, or neither. Combining small EST collections from related species to compensate for their

Proteomics Analysis Reveals Abnormal Electron Transport and Excessive Oxidative Stress Cause Mitochondrial Dysfunction in Placental Tissues of Early-Onset Preeclampsia.

Science.gov (United States)

Xu, Zhongwei; Jin, Xiaohan; Cai, Wei; Zhou, Maobin; Shao, Ping; Yang, Zhen; Fu, Rong; Cao, Jin; Liu, Yan; Yu, Fang; Fan, Rong; Zhang, Yan; Zou, Shuang; Zhou, Xin; Yang, Ning; Chen, Xu; Li, Yuming

2018-04-20

Early-onset preeclampsia (EOS-PE) refers to preeclampsia that occurred before 34 gestation weeks. This study was conducted to explore the relationship between mitochondrial dysfunction and the pathogenesis of EOS-PE using proteomic strategy. To identify altering expressed mitochondrial proteins between severe EOS-PE and healthy pregnancies, enrichment of mitochondria coupled with iTRAQ-based quantitative proteomic method was performed. IHC and western blot were performed to detect the alteration of changing expression proteins, and confirmed the accuracy of proteomic results. We totally quantified 1372 proteins and screened 132 altering expressed mitochondrial proteins, including 86 down-regulated expression proteins and 46 up-regulated expression proteins (pelectron transport chain and oxidative phosphorylation. Especially, mitochondrial related molecules, PRDX2, PARK7, BNIP3, BCL2, PDHA1, SUCLG1, ACADM and NDUFV1, were involved in energy production process in the matrix and membrane of mitochondria. Our results showed that abnormal electron transport, excessive oxidative stress and mitochondrion disassembly might be the main cause of mitochondrial dysfunction, and was related to the pathogenesis of EOS-PE. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Proteomic-based insight into Malpighian tubules of silkworm Bombyx mori.

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Xiao-wu Zhong

Full Text Available Malpighian tubules (MTs are highly specific organs of arthropods (Insecta, Myriapoda and Arachnida for excretion and osmoregulation. In order to highlight the important genes and pathways involved in multi-functions of MTs, we performed a systematic proteomic analysis of silkworm MTs in the present work. Totally, 1,367 proteins were identified by one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry, and as well as by Trans Proteomic Pipeline (TPP and Absolute protein expression (APEX analyses. Forty-one proteins were further identified by two-dimensional gel electrophoresis. Some proteins were revealed to be significantly associated with various metabolic processes, organic solute transport, detoxification and innate immunity. Our results might lay a good foundation for future functional studies of MTs in silkworm and other lepidoptera.

Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

KAUST Repository

Chandramouli, Kondethimmanahalli

2013-03-01

Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

KAUST Repository

Chandramouli, Kondethimmanahalli; Sun, Jin; Mok, FloraSy; Liu, Lingli; Qiu, Jianwen; Ravasi, Timothy; Qian, Peiyuan

2013-01-01

Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings

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Zhujia Ye

2016-08-01

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

Omics strategies for revealing Yersinia pestis virulence

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Yang, Ruifu; Du, Zongmin; Han, Yanping; Zhou, Lei; Song, Yajun; Zhou, Dongsheng; Cui, Yujun

2012-01-01

Omics has remarkably changed the way we investigate and understand life. Omics differs from traditional hypothesis-driven research because it is a discovery-driven approach. Mass datasets produced from omics-based studies require experts from different fields to reveal the salient features behind these data. In this review, we summarize omics-driven studies to reveal the virulence features of Yersinia pestis through genomics, trascriptomics, proteomics, interactomics, etc. These studies serve as foundations for further hypothesis-driven research and help us gain insight into Y. pestis pathogenesis. PMID:23248778

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

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

2011-01-01

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

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

Directory of Open Access Journals (Sweden)

Yu-Hsuan Tu

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

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

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Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

2014-09-15

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

International Nuclear Information System (INIS)

Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

2014-01-01

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa

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Dalia Ponce

2016-04-01

Full Text Available Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa)

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Ponce, Dalia; Brinkman, Diane L.; Potriquet, Jeremy; Mulvenna, Jason

2016-01-01

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms. PMID:27058558

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.

A proteomics study of barley powdery mildew haustoria.

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Godfrey, Dale; Zhang, Ziguo; Saalbach, Gerhard; Thordal-Christensen, Hans

2009-06-01

A number of fungal and oomycete plant pathogens of major economic importance feed on their hosts by means of haustoria, which they place inside living plant cells. The underlying mechanisms are poorly understood, partly due to difficulty in preparing haustoria. We have therefore developed a procedure for isolating haustoria from the barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). We subsequently aimed to understand the molecular mechanisms of haustoria through a study of their proteome. Extracted proteins were digested using trypsin, separated by LC, and analysed by MS/MS. Searches of a custom Bgh EST sequence database and the NCBI-NR fungal protein database, using the MS/MS data, identified 204 haustoria proteins. The majority of the proteins appear to have roles in protein metabolic pathways and biological energy production. Surprisingly, pyruvate decarboxylase (PDC), involved in alcoholic fermentation and commonly abundant in fungi and plants, was absent in our Bgh proteome data set. A sequence encoding this enzyme was also absent in our EST sequence database. Significantly, BLAST searches of the recently available Bgh genome sequence data also failed to identify a sequence encoding this enzyme, strongly indicating that Bgh does not have a gene for PDC.

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.

Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus.

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Zhao, Zelong; Jiang, Jingwei; Pan, Yongjia; Sun, Hongjuan; Guan, Xiaoyan; Gao, Shan; Chen, Zhong; Dong, Ying; Zhou, Zunchun

2018-03-20

Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. The current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these

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

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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.

Single-cell proteomics: potential implications for cancer diagnostics.

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Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

2016-01-01

Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae.

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Kumar, Ravinder; Dhali, Snigdha; Srikanth, Rapole; Ghosh, Santanu Kumar; Srivastava, Sanjeeva

2014-09-23

Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (pmitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

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Lukas Aeberhard

2015-06-01

Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

Impact of urbanization of the proteome of birch pollen and its chemotactic activity on human granulocytes

NARCIS (Netherlands)

Bryce, M.; Drews, O.; Schenk, M.F.; Menzel, A.; Estrella, N.; Weichenmeier, I.; Smulders, M.J.M.; Buters, J.; Ring, J.; Gorg, A.; Behrendt, H.; Traidl-Hoffmann, C.

2010-01-01

Background: Epidemiologic studies reveal a dramatic increase in allergies in the last decades. Air pollution is considered to be one of the factors responsible for this augmentation. The aim of this study was to analyze the impact of urbanization on birch pollen. The birch pollen proteome was

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

Science.gov (United States)

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

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...

iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis.

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Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

2016-01-01

Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

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Naboulsi, Wael; Megger, Dominik A; Bracht, Thilo; Kohl, Michael; Turewicz, Michael; Eisenacher, Martin; Voss, Don Marvin; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

2016-01-04

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani.

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Bhavna Chawla

Full Text Available Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.

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.

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....

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

Science.gov (United States)

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.

Social network architecture of human immune cells unveiled by quantitative proteomics.

Science.gov (United States)

Rieckmann, Jan C; Geiger, Roger; Hornburg, Daniel; Wolf, Tobias; Kveler, Ksenya; Jarrossay, David; Sallusto, Federica; Shen-Orr, Shai S; Lanzavecchia, Antonio; Mann, Matthias; Meissner, Felix

2017-05-01

The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

Centrosome isolation and analysis by mass spectrometry-based proteomics

DEFF Research Database (Denmark)

Jakobsen, Lis; Schrøder, Jacob Morville; Larsen, Katja M

2013-01-01

Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined with advan...... to isolate centrosomes from human cells and strategies to selectively identify and study the properties of the associated proteins using quantitative mass spectrometry-based proteomics.......Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined...... with advances in protein identification using mass spectrometry-based proteomics, have revealed multiple centriole-associated proteins that are conserved during evolution in eukaryotes. Despite these advances, the molecular basis for the plethora of processes coordinated by cilia and centrosomes is not fully...

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

International Nuclear Information System (INIS)

Lo, Andy; Weiner, Joel H.; Li, Liang

2013-01-01

Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

Proteomic and metabolomic approaches to biomarker discovery

CERN Document Server

Issaq, Haleem J

2013-01-01

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

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

Science.gov (United States)

McGorum, Bruce C; Pirie, R Scott; Eaton, Samantha L; Keen, John A; Cumyn, Elizabeth M; Arnott, Danielle M; Chen, Wenzhang; Lamont, Douglas J; Graham, Laura C; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M

2015-11-01

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell

Proteomics in medical microbiology.

Science.gov (United States)

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.

Comparative proteomic analysis of differentially expressed proteins in shoots of Salicornia europaea under different salinity.

Science.gov (United States)

Wang, Xuchu; Fan, Pengxiang; Song, Hongmiao; Chen, Xianyang; Li, Xiaofang; Li, Yinxin

2009-07-01

Soil salinity is a major abiotic stress that limits agriculture productivity worldwide. Salicornia europaea is a succulent annual euhalophyte and one of the most salt tolerant plant species. The elucidation of its salt tolerance mechanism is of significance for generating salt-tolerant crops. In this study, we provided high resolution of proteome reference maps of S. europaea shoot and obtained evidence on the salt tolerance mechanism by analyzing the proteomic responses of this plant to high salinity. Our results demonstrated significant variations existed in 196 out of 1880 protein spots detected on CBB stained 2-DE gels. Of these, 111 proteins were identified by mass spectrometry. Among them, the majority was energy production and conversion related proteins, followed by photosynthesis and carbohydrate metabolism associated enzymes. Analysis of protein expression patters revealed that energy production and ion homeostasis associated proteins played important roles for this plant salt tolerance ability. Hierarchical clustering results revealed many proteins were involved in S. europaea salt tolerance mechanism as a dynamic network. Finally, based on our proteomic results, we brought forward a possible schematic representation of mechanism associated with the systematic salt tolerance phenotype in S. europaea.

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

Proteomics as a tool for studying bacterial virulence and antimicrobial resistance

Directory of Open Access Journals (Sweden)

Francisco José Pérez -Llarena

2016-03-01

Full Text Available Proteomic studies have improved our understanding of the microbial world. The most recent advances in this field have helped us to explore aspects beyond genomics. For example, by studying proteins and their regulation, researchers now understand how some pathogenic bacteria have adapted to the lethal actions of antibiotics. Proteomics has also advanced our knowledge of mechanisms of bacterial virulence and some important aspects of how bacteria interact with human cells and, thus, of the pathogenesis of infectious diseases. This review article addresses these issues in some of the most important human pathogens. It also reports some applications of MALDI-TOF mass spectrometry that may be important for the diagnosis of bacterial resistance in clinical laboratories in the future. The reported advances will enable new diagnostic and therapeutic strategies to be developed in the fight against some of the most lethal bacteria affecting humans.

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Expression proteomics study to determine metallodrug targets and optimal drug combinations.

Science.gov (United States)

Lee, Ronald F S; Chernobrovkin, Alexey; Rutishauser, Dorothea; Allardyce, Claire S; Hacker, David; Johnsson, Kai; Zubarev, Roman A; Dyson, Paul J

2017-05-08

The emerging technique termed functional identification of target by expression proteomics (FITExP) has been shown to identify the key protein targets of anti-cancer drugs. Here, we use this approach to elucidate the proteins involved in the mechanism of action of two ruthenium(II)-based anti-cancer compounds, RAPTA-T and RAPTA-EA in breast cancer cells, revealing significant differences in the proteins upregulated. RAPTA-T causes upregulation of multiple proteins suggesting a broad mechanism of action involving suppression of both metastasis and tumorigenicity. RAPTA-EA bearing a GST inhibiting ethacrynic acid moiety, causes upregulation of mainly oxidative stress related proteins. The approach used in this work could be applied to the prediction of effective drug combinations to test in cancer chemotherapy clinical trials.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves.

Science.gov (United States)

Kieffer, Pol; Planchon, Sébastien; Oufir, Mouhssin; Ziebel, Johanna; Dommes, Jacques; Hoffmann, Lucien; Hausman, Jean-François; Renaut, Jenny

2009-01-01

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.

Modern Biotechnology of Phellinus baumii – From Fermentation to Proteomics

Directory of Open Access Journals (Sweden)

Hye Jin Hwang

2007-01-01

Full Text Available Phellinus baumii is a mushroom used as a folk medicine for a variety of human diseases in several Asian countries. Recently we have reported for the first time about the antidiabetic effect of the crude exopolysaccharides (EPS produced from submerged mycelial culture of P. baumii in streptozotocin (STZ-induced diabetic rats. The diabetic rats study revealed that orally administrated P. baumii EPS lowered the blood glucose levels and stimulated insulin excretion in diabetic rats, and consequently restored the functions of pancreas, liver, and kidney, suggesting that the EPS might be useful for the management of human diabetes mellitus. We undertook proteomic analyses for plasma, pancreas, liver, and kidney of the rats to search for novel biomarkers for monitoring diabetes before and after EPS treatments. In this article, we describe the production of EPS in submerged culture of P. baumii and studies of their hypoglycemic activity. We also explore the issue of proteomic analyses for mining biomarkers of diabetes.

In-Depth Characterization of Sheep (Ovis aries) Milk Whey Proteome and Comparison with Cow (Bos taurus)

Science.gov (United States)

Ha, Minh; Sabherwal, Manya; Duncan, Elizabeth; Stevens, Stewart; Stockwell, Peter; McConnell, Michelle; Bekhit, Alaa El-Din; Carne, Alan

2015-01-01

An in-depth proteomic study of sheep milk whey is reported and compared to the data available in the literature for the cow whey proteome. A combinatorial peptide ligand library kit (ProteoMiner) was used to normalize protein abundance in the sheep whey proteome followed by an in-gel digest of a 1D-PAGE display and an in-solution digestion followed by OFFGEL isoelectric focusing fractionation. The peptide fractions obtained were then analyzed by LC-MS/MS. This enabled identification of 669 proteins in sheep whey that, to our knowledge, is the largest inventory of sheep whey proteins identified to date. A comprehensive list of cow whey proteins currently available in the literature (783 proteins from unique genes) was assembled and compared to the sheep whey proteome data obtained in this study (606 proteins from unique genes). This comparison revealed that while the 233 proteins shared by the two species were significantly enriched for immune and inflammatory responses in gene ontology analysis, proteins only found in sheep whey in this study were identified that take part in both cellular development and immune responses, whereas proteins only found in cow whey in this study were identified to be associated with metabolism and cellular growth. PMID:26447763

In-Depth Characterization of Sheep (Ovis aries Milk Whey Proteome and Comparison with Cow (Bos taurus.

Directory of Open Access Journals (Sweden)

Minh Ha

Full Text Available An in-depth proteomic study of sheep milk whey is reported and compared to the data available in the literature for the cow whey proteome. A combinatorial peptide ligand library kit (ProteoMiner was used to normalize protein abundance in the sheep whey proteome followed by an in-gel digest of a 1D-PAGE display and an in-solution digestion followed by OFFGEL isoelectric focusing fractionation. The peptide fractions obtained were then analyzed by LC-MS/MS. This enabled identification of 669 proteins in sheep whey that, to our knowledge, is the largest inventory of sheep whey proteins identified to date. A comprehensive list of cow whey proteins currently available in the literature (783 proteins from unique genes was assembled and compared to the sheep whey proteome data obtained in this study (606 proteins from unique genes. This comparison revealed that while the 233 proteins shared by the two species were significantly enriched for immune and inflammatory responses in gene ontology analysis, proteins only found in sheep whey in this study were identified that take part in both cellular development and immune responses, whereas proteins only found in cow whey in this study were identified to be associated with metabolism and cellular growth.

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings.

Science.gov (United States)

Sang, Ting; Shan, Xi; Li, Bin; Shu, Sheng; Sun, Jin; Guo, Shirong

2016-08-01

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings. Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

Diversification of the muscle proteome through alternative splicing.

Science.gov (United States)

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

Differential proteomics reveals novel insights into Nosema-honey bee interactions.

Science.gov (United States)

Kurze, Christoph; Dosselli, Ryan; Grassl, Julia; Le Conte, Yves; Kryger, Per; Baer, Boris; Moritz, Robin F A

2016-12-01

Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteomic analysis of Taenia hydatigena cyst fluid reveals unique internal microenvironment.

Science.gov (United States)

Zheng, Yadong

2017-12-01

Taenia hydatigena is a parasitic flatworm that is widely distributed around the world. Using MS/MS, the proteome of T. hydatigena cyst fluid (CF) was profiled and a total of 520 proteins were identified, 430 of which were of sheep origin. T. hydatigena shared 37 parasite-origin and 109 host-origin CF proteins with Echinococcus granulosus. Compared with E. granulosus, T. hydatigena had much more CF proteins associated with amino acid synthesis and complement cascades. In addition, glutamate metabolism and anti-oxidative reactions were identified as relatively more important events. These results suggest that T. hydatigena metacestodes have internal microenvironment with special immune and oxidative conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative proteome-level analysis of paulownia witches’ broom disease with methyl methane sulfonate assistance reveals diverse metabolic changes during the infection and recovery processes

Directory of Open Access Journals (Sweden)

Zhe Wang

2017-07-01

Full Text Available Paulownia witches’ broom (PaWB disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ to quantify and analyze the proteome-level changes among healthy P. fortunei (PF, PaWB-infected P. fortunei (PFI, and PaWB-infected P. fortunei treated with 20 mg L−1 or 60 mg L−1 methyl methane sulfonate (MMS (PFI-20 and PFI-60, respectively. A total of 2,358 proteins were identified. We investigated the proteins profiles in PF vs. PFI (infected process and PFI-20 vs. PFI-60 (recovered process, and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1 carbohydrate and energy metabolism, (2 protein synthesis and degradation, and (3 stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

Análise proteômica da esquizofrenia Large-scale analyses of schizophrenia proteome

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Bruno M. Oliveira

2013-01-01

Full Text Available Valioso conhecimento a respeito de esquizofrenia tem sido gerado recentemente para decifrar sua patobiologia e revelar biomarcadores. Entretanto, esforços ainda são necessários, especialmente se levarmos em conta que essa debilitante desordem mental afeta aproximadamente 30 milhões de pessoas ao redor do mundo. Considerando que esquizofrenia é resultado de uma complexa interação entre fatores ambientais, função genética alterada e expressão proteica diferencial sistemática, a proteômica é provavelmente uma ferramenta adequada ao estudo dessa desordem. Aqui sintetizamos os principais achados em estudos proteômicos e posteriores direções a serem tomadas de forma a melhor compreender a bioquímica da esquizofrenia, bem como revelar biomarcadores.Valuable knowledge about schizophrenia has been recently generated for deciphering its pathobiology and revealing biomarkers. However, efforts are still needed, especially if we take in account that this debilitating mental disorder affects approximately 30 million people worldwide. Considering that schizophrenia is a result of a complex interaction among environmental factors altered gene function and systematic differential protein expression, proteomics is likely to be a suitable tool for studying this disorder. Here we synthetize the main findings by proteomic studies and further directions to be taken in order to better comprehend the biochemistry of schizophrenia as well as reveal biomarkers. In addition, we summarize proteomic methodologies used in such studies.

Análise proteômica da esquizofrenia Large-scale analyses of schizophrenia proteome

Directory of Open Access Journals (Sweden)

M. Oliveira

2012-01-01

Full Text Available Valioso conhecimento a respeito de esquizofrenia tem sido gerado recentemente para decifrar sua patobiologia e revelar biomarcadores. Entretanto, esforços ainda são necessários, especialmente se levarmos em conta que essa debilitante desordem mental afeta aproximadamente 30 milhões de pessoas ao redor do mundo. Considerando que esquizofrenia é resultado de uma complexa interação entre fatores ambientais, função genética alterada e expressão proteica diferencial sistemática, a proteômica é provavelmente uma ferramenta adequada ao estudo dessa desordem. Aqui sintetizamos os principais achados em estudos proteômicos e posteriores direções a serem tomadas de forma a melhor compreender a bioquímica da esquizofrenia, bem como revelar biomarcadores.Valuable knowledge about schizophrenia has been recently generated for deciphering its pathobiology and revealing biomarkers. However, efforts are still needed, especially if we take in account that this debilitating mental disorder affects approximately 30 million people worldwide. Considering that schizophrenia is a result of a complex interaction among environmental factors altered gene function and systematic differential protein expression, proteomics is likely to be a suitable tool for studying this disorder. Here we synthetize the main findings by proteomic studies and further directions to be taken in order to better comprehend the biochemistry of schizophrenia as well as reveal biomarkers. In addition, we summarize proteomic methodologies used in such studies.

Fusarium graminearum and Its Interactions with Cereal Heads: Studies in the Proteomics Era

DEFF Research Database (Denmark)

Yang, Fen; Jacobsen, Susanne; Jørgensen, Hans J L

2013-01-01

of humans and animals. In recent years, high-throughput proteomics, aiming at identifying a broad spectrum of proteins with a potential role in the pathogenicity and host resistance, has become a very useful tool in plant-fungus interaction research. In this review, we describe the progress in proteomics...... applications toward a better understanding of pathogenesis, virulence, and host defense mechanisms. The contribution of proteomics to the development of crop protection strategies against this pathogen is also discussed briefly....

Dissecting the chloroplast proteome of chickpea (Cicer arietinum L.) provides new insights into classical and non-classical functions.

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Lande, Nilesh Vikram; Subba, Pratigya; Barua, Pragya; Gayen, Dipak; Keshava Prasad, T S; Chakraborty, Subhra; Chakraborty, Niranjan

2017-08-08

Chloroplast, the energy organelle unique to plant cells, is a dynamic entity which integrates an array of metabolic pathways and serves as first level for energy conversion for the entire ecological hierarchy. Increasing amount of sequence data and evolution of mass spectrometric approaches has opened up new avenues for opportune exploration of the global proteome of this organelle. In our study, we aimed at generation of a comprehensive catalogue of chloroplast proteins in a grain legume, chickpea and provided a reference proteome map. To accurately assign the identified proteins, purity of chloroplast-enriched fraction was stringently monitored by multiple chemical and immunological indexes, besides pigment and enzyme analyses. The proteome analysis led to the identification of 2451 proteins, including 27 isoforms, which include predicted and novel chloroplast constituents. The identified proteins were validated through their sequence analysis. Extensive sequence based localization prediction revealed more than 50% proteins to be chloroplast resident by at least two different algorithms. Chromosomal distribution of identified proteins across nuclear and chloroplast genome unveiled the presence of 55 chloroplast encoded gene. In depth comparison of our dataset with the non-redundant set of chloroplast proteins identified so far across other species revealed novel as well as overlapping candidates. Pulses add large amount of nitrogen to the soil and has very low water footprint and therefore, contributes to fortification of sustainable agriculture. Chickpea is one of the earliest cultivated legumes and serves as an energy and protein source for humans and animals. Chloroplasts are the unique organelles which conduct photosynthesis. Investigation on chloroplast proteome is of particular significance, especially to plant biologists, as it would allow a better understanding of chloroplast function in plants. Generation of a saturated proteome map would not only

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.

Proteomics and plant disease: advances in combating a major threat to the global food supply.

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Rampitsch, Christof; Bykova, Natalia V

2012-02-01

The study of plant disease and immunity is benefiting tremendously from proteomics. Parallel streams of research from model systems, from pathogens in vitro and from the relevant pathogen-crop interactions themselves have begun to reveal a model of how plants succumb to invading pathogens and how they defend themselves without the benefit of a circulating immune system. In this review, we discuss the contribution of proteomics to these advances, drawing mainly on examples from crop-fungus interactions, from Arabidopsis-bacteria interactions, from elicitor-based model systems and from pathogen studies, to highlight also the important contribution of non-crop systems to advancing crop protection. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis of tissue from α1,3-galactosyltransferase knockout mice reveals that a wide variety of proteins and protein fragments change expression level.

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Louise Thorlacius-Ussing

Full Text Available A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient's blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes.

Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development.

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Wang, Yong-Qiang; Yang, Yong; Fei, Zhangjun; Yuan, Hui; Fish, Tara; Thannhauser, Theodore W; Mazourek, Michael; Kochian, Leon V; Wang, Xiaowu; Li, Li

2013-02-01

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953-2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.

Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile

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Vilma Pérez

2017-06-01

Full Text Available Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l., permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps, cut-off filters (Mylar, Lee filters and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins.

Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

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Da-Zhi Wang

2013-01-01

Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

Proteomic analysis of albumin and globulin fractions of pea (Pisum sativum L.) seeds.

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Dziuba, Jerzy; Szerszunowicz, Iwona; Nałęcz, Dorota; Dziuba, Marta

2014-01-01

Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thousands of proteins. In this study, albumin and globulin tractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenic proteins were identified based on their molecular weights and isoelectric points. Pea seeds (Pisum sativum L.) cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo) were used in the experiment. The isolated albumins, globulins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identified proteins. Electrophoregrams were analysed by matching spot positions from three independent replications. The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins). Spots most characteristic of a given fraction were identified by computer analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pi values of ~4.2-8.1 in 3 molecular weight (MW) ranges: I - high molecular-weight albumins with MW of ~50-110 kDa, II - average molecular-weight albumins with MW of ~20-35 kDa, and III - low molecular-weight albumins with MW of ~13-17 kDa. 2D gel electrophoregrams revealed the presence of 81 characteristic spots, including 24 characteristic of legumin and 14 - of vicilin. Two-dimensional gel electrophoresis proved to be a useful tool for identifying pea proteins. Patterns of spots with similar isoelectric

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio.

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Ozlem Yilmaz

Full Text Available Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio).

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Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Translational plant proteomics: a perspective.

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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.

Oxidative Stress in Aging: Advances in Proteomic Approaches

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Daniel Ortuño-Sahagún

2014-01-01

Full Text Available Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual’s Quality of Life (QOL. Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS], which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8, naked mole-rat (Heterocephalus glaber, and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS and oxidative stress in aging.

Proteomic approach in human health and disease: Preventive and cure studies

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Khaled MM Koriem

2018-01-01

Full Text Available Proteomic is a branch of science that deals with various numbers of proteins where proteins are essential human constituents. Proteomic has a lot of functions inside the human and animal living organisms. This review helps to make a thought on the importance of proteomic application in human health and disease with special reference to preventive and cure studies. The human health can be divided into physical and mental health. The physical health relates to keeping human body state in a good health and to nutritional type and environmental factors. The mental health correlates to human psychological state. The main factors that affect the status of human health are human diet, exercise and sleep. The healthy diet is very important and needs to maintain the human health. The training program exercise improves human fitness and overall health and wellness. The sleep is a vital factor to sustain the human health. The human disease indicates abnormal human condition which influences the specific human part or the whole human body. There are external and internal factors which induce human disease. The external factors include pathogens while internal factors include allergies and autoimmunity. There are 4 principle types of human diseases: (1 infectious disease, (2 deficiency disease, (3 genetic disease and (4 physiological disease. There are many and various external microbes' factors that induce human infectious disease and these agents include viruses, bacteria, fungi and protozoa. The lack of necessary and vital dietary rudiments such as vitamins and minerals is the main cause of human deficiency disease. The genetic disease is initiated by hereditary disturbances that occur in the human genetic map. The physiological disease occurs when the normal human function body is affected due to human organs become malfunction. In conclusion, proteomic plays a vital and significant role in human health and disease.

Spermatogenesis in mammals: proteomic insights.

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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

Quantitative proteomic analysis of post-translational modifications of human histones

DEFF Research Database (Denmark)

Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune

2006-01-01

, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational...

Alteration of cystatin C in cerebrospinal fluid of patients with sciatica revealed by a proteomical approach

International Nuclear Information System (INIS)

Liu, X; Zeng, B.; Xu, J.

2005-01-01

To better understand the pathophysiological mechanisms underlying sciatica induced by lumbar intervertebral disk herniation and to ascertain the protein that presents with the most observable changes in the cerebrospinal fluid (CSF) of patients with sciatica. We conducted the study in the Key Laboratory of Shanghai 6th People's Hospital, Shanghai Jiaotong University, Shanghai, Peoples Republic of China, during the period June 2004 to March 2005. In 2 separate experiments, we carried out the study involving the CSF of sciatica patients (the case group) and the CSF of otherwise, healthy volunteers (the control group). We utilized a proteomical analysis to compare the samples of 10 patients with sciatica with 10 volunteers in the control group. We individually separated each of the groups' CSF by 2-dimensional gel electrophoresis. We analyzed the harvested gel images with PD Quest 2D-gel software (Bio-Rad) to ascertain the differential proteins between the 2 groups. We based the enzyme linked immuno- absorbent assay (ELISA) experiment, which followed, on the results of the first experiment. We found 15 of the protein spots in the CSF differed appreciably in varying degrees between the 2 groups, and identification made by LC-MS/MS revealed that the most significant disparity was with cystatin C. The result of ELISA experiment confirmed a considerable decrease in the level of cystatin C (p<0.01) in the patients with sciatica. In the CSF of patients with sciatica, the volume of cystatin C increased markedly indicating that it may play an important role in the pathophysiological processes of sciatica. (author)

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.

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

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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.

Comprehensive proteomic analysis of human pancreatic juice

DEFF Research Database (Denmark)

Grønborg, Mads; Bunkenborg, Jakob; Kristiansen, Troels Zakarias

2004-01-01

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity...... contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid...... in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays....

Characterizing the proteome and oxi-proteome of apple in response to a host (Penicillium expansum) and a non-host (Penicillium digitatum) pathogen.

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Buron-Moles, Gemma; Wisniewski, Michael; Viñas, Inmaculada; Teixidó, Neus; Usall, Josep; Droby, Samir; Torres, Rosario

2015-01-30

of chemical fungicides and the implementation of new alternative strategies, blue mold remains a critical disease of these stored fruits worldwide. Actual trends are focused on acquiring the knowledge of the host-pathogen interactions because it may help on finding new rational and environmentally friendly control alternatives. Despite the economic importance of some postharvest diseases, proteomics has only been applied in a few cases to study fruit-pathogen interactions. On the one hand, this is the first study that monitored changes at the proteome and oxi-proteome level in 'Golden Smoothee' apple fruits in response to P. expansum (compatible) and P. digitatum (non-host) pathogens. On the other hand, the main technological innovation of the reported research is the detection and quantification of oxidized (carbonylated) proteins to assess protein oxidative damage, avoiding the immunoblotting technique. The importance of the biological process investigated lies in the different mechanisms induced in fruit in response to P. expansum and P. digitatum. Results revealed that fruit recognizes and reacts to P. expansum in a similar manner to wounding, while its response to P. digitatum exhibits few differences in the protein profile. Documenting changes in the proteome and, specifically in oxi-proteome of apple can provide information that can be used to better understand how impaired protein functions may affect apple defense mechanisms. It also provides new biomarkers for oxidative damage mainly caused by the oxidative response occurring in fruit tissue in response to a host and a non-host pathogen. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics: Protein Identification Using Online Databases

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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...

Evolution of proteomes: fundamental signatures and global trends in amino acid compositions

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Yeramian Edouard

2006-12-01

Full Text Available Abstract Background The evolutionary characterization of species and lifestyles at global levels is nowadays a subject of considerable interest, particularly with the availability of many complete genomes. Are there specific properties associated with lifestyles and phylogenies? What are the underlying evolutionary trends? One of the simplest analyses to address such questions concerns characterization of proteomes at the amino acids composition level. Results In this work, amino acid compositions of a large set of 208 proteomes, with significant number of representatives from the three phylogenetic domains and different lifestyles are analyzed, resorting to an appropriate multidimensional method: Correspondence analysis. The analysis reveals striking discrimination between eukaryotes, prokaryotic mesophiles and hyperthemophiles-themophiles, following amino acid usage. In sharp contrast, no similar discrimination is observed for psychrophiles. The observed distributional properties are compared with various inferred chronologies for the recruitment of amino acids into the genetic code. Such comparisons reveal correlations between the observed segregations of species following amino acid usage, and the separation of amino acids following early or late recruitment. Conclusion A simple description of proteomes according to amino acid compositions reveals striking signatures, with sharp segregations or on the contrary non-discriminations following phylogenies and lifestyles. The distribution of species, following amino acid usage, exhibits a discrimination between [high GC]-[high optimal growth temperatures] and [low GC]-[moderate temperatures] characteristics. This discrimination appears to coincide closely with the separation of amino acids following their inferred early or late recruitment into the genetic code. Taken together the various results provide a consistent picture for the evolution of proteomes, in terms of amino acid usage.

The Seminal fluid proteome of the polyandrous Red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication.

Science.gov (United States)

Borziak, Kirill; Álvarez-Fernández, Aitor; L Karr, Timothy; Pizzari, Tommaso; Dorus, Steve

2016-11-02

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.

Comparative proteomic analysis of lung tissue from guinea pigs with Leptospiral Pulmonary Haemorrhage Syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

Science.gov (United States)

The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, a 2-D guinea pig proteome lung map was used to investigate the pathogenic mechanisms of ...

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....

Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

DEFF Research Database (Denmark)

Hendriks, Ivo A; Lyon, David; Young, Clifford

2017-01-01

that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

Comparative proteomic and metabolomic analysis of Streptomyces tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil.

Science.gov (United States)

Wang, Jun; Liu, Huanhuan; Huang, Di; Jin, Lina; Wang, Cheng; Wen, Jianping

2017-03-01

FK506 (tacrolimus) is a 23-membered polyketide macrolide that possesses powerful immunosuppressant activity. In this study, feeding soybean oil into the fermentation culture of Streptomyces tsukubaensis improved FK506 production by 88.8%. To decipher the overproduction mechanism, comparative proteomic and metabolomic analysis was carried out. A total of 72 protein spots with differential expression in the two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), and 66 intracellular metabolites were measured by gas chromatography-mass spectrometer (GC-MS). The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation, translation, and stress response, suggesting a better intracellular metabolic environment for the synthesis of FK506. Based on these analyses, 20 key metabolites and precursors of FK506 were supplemented into the soybean oil medium. Among them, lysine, citric acid, shikimic acid, and malonic acid performed excellently for promoting the FK506 production and biomass. Especially, the addition of malonic acid achieved the highest FK506 production, which was 1.56-fold of that in soybean oil medium and 3.05-fold of that in initial medium. This report represented the first comprehensive study on the comparative proteomics and metabolomics applied in S. tsukubaensis, and it would be a rational guidance to further strengthen the FK506 production.

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

The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse phosphorus stress response.

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Sonya T Dyhrman

Full Text Available Phosphorus (P is a critical driver of phytoplankton growth and ecosystem function in the ocean. Diatoms are an abundant class of marine phytoplankton that are responsible for significant amounts of primary production. With the control they exert on the oceanic carbon cycle, there have been a number of studies focused on how diatoms respond to limiting macro and micronutrients such as iron and nitrogen. However, diatom physiological responses to P deficiency are poorly understood. Here, we couple deep sequencing of transcript tags and quantitative proteomics to analyze the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. A total of 318 transcripts were differentially regulated with a false discovery rate of <0.05, and a total of 136 proteins were differentially abundant (p<0.05. Significant changes in the abundance of transcripts and proteins were observed and coordinated for multiple biochemical pathways, including glycolysis and translation. Patterns in transcript and protein abundance were also linked to physiological changes in cellular P distributions, and enzyme activities. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P through increased production of metalloenzymes, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to respond to variations in environmental P availability.

Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

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Yanlong Wang

Full Text Available We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli, Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology and KEGG (Kyoto encyclopedia of genes and genomes enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species scavenging, membrane proteins and ABC (ATP binding cassette transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages.

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Wang, Jing; Gu, Yong; Wang, Lihong; Hang, Xingyi; Gao, Yan; Wang, Hangyan; Zhang, Chenggang

2007-11-01

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.

Revealing proteins associated with symbiotic germination of Gastrodia elata by proteomic analysis.

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Zeng, Xu; Li, Yuanyuan; Ling, Hong; Chen, Juan; Guo, Shunxing

2018-03-06

Gastrodia elata, a mycoheterotrophic orchid, is a well-known medicinal herb. In nature, the seed germination of G. elata requires proper fungal association, because of the absence of endosperm. To germinate successfully, G. elata obtains nutrition from mycorrhizal fungi such as Mycena. However, Mycena is not able to supply nutrition for the further development and enlargement of protocorms into tubers, flowering and fruit setting of G. elata. To date, current genomic studies on this topic are limited. Here we used the proteomic approach to explore changes in G. elata at different stages of symbiotic germination. Using mass spectrometry, 3787 unique proteins were identified, of which 599 were classified as differentially accumulated proteins. Most of these differentially accumulated proteins were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Among them, the defense genes (e.g., pathogenesis-/wound-related proteins, peroxidases, and serine/threonine-protein kinase) were highly expressed in late-stage protocorms, suggesting that fungal colonization triggered the significant defense responses of G. elata. The present study indicated the metabolic change and defensive reaction could disrupt the balance between Mycena and G. elata during mycorrhizal symbiotic germination.

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.

Proteomic profiling of the human T-cell nucleolus.

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Jarboui, Mohamed Ali; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

2011-12-01

The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

Synergistic effects of retinoic acid and tamoxifen on human breast cancer cells: Proteomic characterization

International Nuclear Information System (INIS)

Wang Ying; He Qingyu; Chen Hongming; Chiu Jenfu

2007-01-01

The anti-estrogen tamoxifen and vitamin A-related compound, all-trans retinoic acid (RA), in combination act synergistically to inhibit the growth of MCF-7 human breast cancer cells. In the present study, we applied two-dimensional gel electrophoresis based proteomic approach to globally analyze this synergistic effect of RA and tamoxifen. Proteomic study revealed that multiple clusters of proteins were involved in RA and tamoxifen-induced apoptosis in MCF-7 breast cancer cells, including post-transcriptional and splicing factors, proteins related to cellular proliferation or differentiation, and proteins related to energy production and internal degradation systems. The negative growth factor-transforming growth factor β (TGFβ) was secreted by RA and/or tamoxifen treatment and was studies as a potential mediator of the synergistic effects of RA and tamoxifen in apoptosis. By comparing protein alterations in treatments of RA and tamoxifen alone or in combination to those of TGFβ treatment, or co-treatment with TGFβ inhibitor SB 431542, proteomic results showed that a number of proteins were involved in TGFβ signaling pathway. These results provide valuable insights into the mechanisms of RA and tamoxifen-induced TGFβ signaling pathway in breast cancer cells

[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.

Final Report: Proteomic study of brassinosteroid responses in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Wang, Zhiyong [Carnegie Inst. of Washington, Argonne, IL (United States); Burlingame, Alma [Univ. of California, San Francisco, CA (United States)

2017-11-29

The steroid hormone brassinosteroid (BR) is a major growth-promoting phytohormone. The specific aim of the current project is to identify BR-regulated proteins and characterize their functions in various aspects of plant growth, development, and adaptation. Our research has significantly advanced our understanding of how BR signal is transduced from the receptor at the cell surface to changes of nuclear gene expression and other cellular responses such as vesicle trafficking, as well as developmental transitions such as seed germination and flowering. We have also developed effective proteomic methods for quantitative analysis of protein phosphorylation and for identification of glycosylated proteins. Through this DOE funding, we have performed several proteomic experiments and made major discoveries.

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

Science.gov (United States)

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

2015-01-01

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

Redox proteomic analysis of the gastrocnemius muscle from adult and old mice

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Brian McDonagh

2015-09-01

Full Text Available The data provides information in support of the research article, “Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging”, Journal of Proteome Research, 2014, 13 (11, 2008–21 [1]. Raw data is available from ProteomeXchange [2] with identifier PDX001054. The proteome of gastrocnemius muscle from adult and old mice was analyzed by global label-free proteomics and the relative quantification of specific reduced and reversibly oxidized Cysteine (Cys residues was performed using Skyline [3]. Briefly, reduced Cysteine (Cys containing peptides was alkylated using N-ethylmalemide (d0-NEM. Samples were desalted and reversibly oxidized Cys residues were reduced using tris(2-carboxyethylphosphine (TCEP and the newly formed reduced Cys residues were labeled with heavy NEM( d5-NEM. Label-free analysis of the global proteome of adult (n=5 and old (n=4 gastrocnemius muscles was performed using Peaks7™ mass spectrometry data analysis software [4]. Relative quantification of Cys containing peptides that were identified as reduced (d(0 NEM labeled and reversibly oxidized d(5–NEM labeled was performed using the intensity of their precursor ions in Skyline. Results indicate that muscles from old mice show reduced redox flexibility particularly in proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

Science.gov (United States)

Blundon, Malachi A; Schlesinger, Danielle R; Parthasarathy, Amritha; Smith, Samantha L; Kolev, Hannah M; Vinson, David A; Kunttas-Tatli, Ezgi; McCartney, Brooke M; Minden, Jonathan S

2016-07-15

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development. © 2016. Published by The Company of Biologists Ltd.

Quantitative proteomics revealed partial fungistatic mechanism of ammonia against conidial germination of nematode-trapping fungus Arthrobotrys oligospora ATCC24927.

Science.gov (United States)

Liu, Tong; Tian, Dong-Wei; Zou, Li-Juan; Liu, Fang-Yu; Can, Qi-Yan; Yang, Jin-Kui; Xu, Jian-Ping; Huang, Xiao-Wei; Xi, Jia-Qin; Zhu, Ming-Liang; Mo, Ming-He; Zhang, Ke-Qin

2018-05-01

Ammonia is one of the fungistatic factors in soil that can suppress conidial germination, but the molecular mechanism underlying the suppression is unknown. In this study, the proteomes of fungistatic conidia, fresh conidia and germinated conidia of Arthrobotrys oligospora ATCC24927 were determined and quantified. The protein expression profile of fungistatic conidia was significantly different from those in the other two conditions. 281 proteins were down expressed in fungistatic conidia and characterized by GO annotation. Gene transcription analysis and inhibition of puromycin (a protein translation inhibitor) on conidial germination suggested that down expression of 33 protein translation related proteins might well result in repression of protein synthesis and inhibition of conidial germination. In addition, 16 down-expressed proteins were mapped to the Ras/mitogen-activated protein (Ras/MAP) regulatory networks which regulate conidial DNA synthesis. The conidial DNA synthesis was found to be definitely inhibited under by ammonia, and function studies of two Ras/MAP proteins by using knock-out strains provided partial evidence that Ras/MAP pathway regulate the conidial germination. These results suggested that down-expression of Ras/MAP related proteins might result in inhibition of DNA synthesis and finally result in inhibition conidial germination. This study revealed partial fungistatic mechanism of ammonia against conidial germination. Copyright © 2018 Elsevier Ltd. All rights reserved.

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....

A proteomic and ultrastructural characterization of Aspergillus fumigatus' conidia adaptation at different culture ages.

Science.gov (United States)

Anjo, Sandra I; Figueiredo, Francisco; Fernandes, Rui; Manadas, Bruno; Oliveira, Manuela

2017-05-24

The airborne fungus Aspergillus fumigatus is one of the most common agents of human fungal infections with a remarkable impact on public health. However, A. fumigatus conidia atmospheric resistance and longevity mechanisms are still unknown. Therefore, in this work, the processes underlying conidial adaptation were studied by a time course evaluation of the proteomics and ultrastructural changes of A. fumigatus' conidia at three time-points selected according to relevant changes previously established in conidial survival rates. The proteomics characterization revealed that conidia change from a highly active metabolic to a dormant state, culminating in cell autolysis as revealed by the increased levels of hydrolytic enzymes. Structural characterization corroborates the proteomics data, with noticeable changes observed in mitochondria, nucleus and plasma membrane ultrastructure, accompanied by the formation of autophagic vacuoles. These changes are consistent with both apoptotic and autophagic processes, and indicate that the changes in protein levels may anticipate those in cell morphology. The findings presented in this work not only clarify the processes underlying conidial adaptation to nutrient limiting conditions but can also be exploited for improving infection control strategies and in the development of new therapeutical drugs. Additionally, the present study was deposited in a public database and thus, it may also be a valuable dataset to be used by the scientific community as a tool to understand and identified other potential targets associated with conidia resistance. Copyright © 2017. Published by Elsevier B.V.

Global analysis of the yeast osmotic stress response by quantitative proteomics

DEFF Research Database (Denmark)

Soufi, Boumediene; Kelstrup, C.D.; Stoehr, G.

2009-01-01

a comprehensive, quantitative, and time-resolved analysis using high-resolution mass spectrometry of phospho-proteome and proteome changes in response to osmotic stress in yeast. We identified 5534 unique phosphopeptide variants and 3383 yeast proteins. More than 15% of the detected phosphorylation site status...... changed more than two-fold within 5 minutes of treatment. Many of the corresponding phosphoproteins are involved in the early response to environmental stress. Surprisingly, we find that 158 regulated phosphorylation sites are potential substrates of basophilic kinases as opposed to the classical proline......-directed MAP kinase network implicated in stress response mechanisms such as p38 and HOG pathways. Proteome changes reveal an increase in abundance of more than one hundred proteins after 20 min of salt stress. Many of these are involved in the cellular response to increased osmolarity, which include proteins...

Mass Spectrometry-Based Proteomic Profiling of Thrombotic Material Obtained by Endovascular Thrombectomy in Patients with Ischemic Stroke

Directory of Open Access Journals (Sweden)

Roberto Muñoz

2018-02-01

Full Text Available Thrombotic material retrieved from acute ischemic stroke (AIS patients represents a valuable source of biological information. In this study, we have developed a clinical proteomics workflow to characterize the protein cargo of thrombi derived from AIS patients. To analyze the thrombus proteome in a large-scale format, we developed a workflow that combines the isolation of thrombus by endovascular thrombectomy and peptide chromatographic fractionation coupled to mass-spectrometry. Using this workflow, we have characterized a specific proteomic expression profile derived from four AIS patients included in this study. Around 1600 protein species were unambiguously identified in the analyzed material. Functional bioinformatics analyses were performed, emphasizing a clustering of proteins with immunological functions as well as cardiopathy-related proteins with blood-cell dependent functions and peripheral vascular processes. In addition, we established a reference proteomic fingerprint of 341 proteins commonly detected in all patients. Protein interactome network of this subproteome revealed protein clusters involved in the interaction of fibronectin with 14-3-3 proteins, TGFβ signaling, and TCP complex network. Taken together, our data contributes to the repertoire of the human thrombus proteome, serving as a reference library to increase our knowledge about the molecular basis of thrombus derived from AIS patients, paving the way toward the establishment of a quantitative approach necessary to detect and characterize potential novel biomarkers in the stroke field.

Virtual Labs in proteomics: new E-learning tools.

Science.gov (United States)

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.

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.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

Science.gov (United States)

Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

2017-05-01

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

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Bianco, Linda; Perrotta, Gaetano

2015-01-01

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation. PMID:25775160

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.

Methodologies and perspectives of proteomics applied to filamentous fungi: from sample preparation to secretome analysis.

Science.gov (United States)

Bianco, Linda; Perrotta, Gaetano

2015-03-12

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.

Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage

International Nuclear Information System (INIS)

Bouthier de la Tour, Claire; Passot, Fanny Marie; Toueille, Magali; Servant, Pascale; Sommer, Suzanne; Mirabella, Boris; Blanchard, Laurence; Groot, Arjan de; Guerin, Philippe; Armengaud, Jean

2013-01-01

The nucleoids of radiation-resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid-enriched fractions from unirradiated and irradiated Deinococcus were identified and semi quantified by shotgun proteomics. The ssDNA-binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament-like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post-irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196. (authors)

The Path to Enlightenment: Making Sense of Genomic and Proteomic Information

OpenAIRE

Maurer, Martin H.

2016-01-01

Whereas genomics describes the study of genome, mainly represented by its gene expression on the DNA or RNA level, the term proteomics denotes the study of the proteome, which is the protein complement encoded by the genome. In recent years, the number of proteomic experiments increased tremendously. While all fields of proteomics have made major technological advances, the biggest step was seen in bioinformatics. Biological information management relies on sequence and structure databases an...

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

Science.gov (United States)

George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

2015-09-01

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

Science.gov (United States)

Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Núñez, Félix; Asensio, Miguel A

2017-09-01

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl 2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl 2 -incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl 2 . Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondrial Molecular Pathophysiology of Nonalcoholic Fatty Liver Disease: A Proteomics Approach

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Natalia Nuño-Lámbarri

2016-03-01

Full Text Available Nonalcoholic fatty liver disease (NAFLD is a chronic liver condition that can progress to nonalcoholic steatohepatitis, cirrhosis and cancer. It is considered an emerging health problem due to malnourishment or a high-fat diet (HFD intake, which is observed worldwide. It is well known that the hepatocytes’ apoptosis phenomenon is one of the most important features of NAFLD. Thus, this review focuses on revealing, through a proteomics approach, the complex network of protein interactions that promote fibrosis, liver cell stress, and apoptosis. According to different types of in vitro and murine models, it has been found that oxidative/nitrative protein stress leads to mitochondrial dysfunction, which plays a major role in stimulating NAFLD damage. Human studies have revealed the importance of novel biomarkers, such as retinol-binding protein 4, lumican, transgelin 2 and hemoglobin, which have a significant role in the disease. The post-genome era has brought proteomics technology, which allows the determination of molecular pathogenesis in NAFLD. This has led to the search for biomarkers which improve early diagnosis and optimal treatment and which may effectively prevent fatal consequences such as cirrhosis or cancer.

Proteomics and circadian rhythms: It’s all about signaling!

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Mauvoisin, Daniel; Dayon, Loïc; Gachon, Frédéric; Kussmann, Martin

2014-01-01

1. Abstract Proteomic technologies using mass spectrometry (MS) offer new perspectives in circadian biology, in particular the possibility to study posttranslational modifications (PTMs). To date, only very few studies have been carried out to decipher the rhythmicity of protein expression in mammals with large-scale proteomics. Although signaling has been shown to be of high relevance, comprehensive characterization studies of PTMs are even more rare. This review aims at describing the actual landscape of circadian proteomics and the opportunities and challenges appearing on the horizon. Emphasis was given to signaling processes for their role in metabolic heath as regulated by circadian clocks and environmental factors. Those signaling processes are expected to be better and more deeply characterized in the coming years with proteomics. PMID:25103677

Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media.

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Stephanie Val

Full Text Available Chronic Otitis Media (COM is characterized by middle ear effusion (MEE and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited.This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses.MEEs were collected from children (n = 49 with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis.109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin.Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions.

Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

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Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

2016-01-04

The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

Proteomic responses reveal the differential effects induced by cadmium in mussels Mytilus galloprovincialis at early life stages.

Science.gov (United States)

Xu, Lanlan; Peng, Xiao; Yu, Deliang; Ji, Chenglong; Zhao, Jianmin; Wu, Huifeng

2016-08-01

Cadmium (Cd) has become an important metal contaminant and posed severe risk on the organisms in the coastal environments of the Bohai Sea. Marine mussel Mytilus galloprovincialis is widely distributed along the Bohai coast and consumed as seafood by local residents. Evidences indicate that the early stages of marine organisms are more sensitive to metal contaminants. In this study, we applied two-dimensional electrophoresis-based proteomics to characterize the biological effects of Cd (50 μg L(-1)) in the early life stages (D-shape larval and juvenile) of mussels. The different proteomic responses demonstrated the differential responsive mechanisms to Cd exposure in these two early life stages of mussels. In details, results indicated that Cd mainly induced immune and oxidative stresses in both D-shape larval and juvenile mussels via different pathways. In addition, the significant up-regulation of triosephosphate isomerase and metallothionein confirmed the enhanced energy demand and mobilized detoxification mechanism in D-shape larval mussels exposed to Cd. In juvenile mussels, Cd exposure also induced clear apoptosis. Overall, this work suggests that Cd is a potential immune toxicant to mussel M. galloprovincialis at early life stages. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Pre-fractionation strategies to resolve pea (Pisum sativum sub-proteomes

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Claudia Nicole Meisrimler

2015-10-01

Full Text Available Legumes are important crop plants and pea (Pisum sativum L. has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula G. allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins. Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

From genomes to vaccines via the proteome

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R Alan Wilson

2004-08-01

Full Text Available An effective vaccine against schistosomiasis mansoni would be a valuable control tool and the high levels of protection elicited in rodents and primates by radiation-attenuated cercariae provide proof of principle. A major obstacle to vaccine development is the difficulty of identifying the antigens that mediate protection, not least because of the size of the genome at 280Mb DNA encoding 14,000 to 20,000 genes. The technologies collectively called proteomics, including 2D electrophoresis, liquid chromatography and mass spectrometry, now permit any protein to be identified provided there is extensive DNA data, and preferably a genome sequence. Applied to soluble (cytosolic proteins from schistosomes, proteomics reveals the great similarity in composition between life cycle stages, with several WHO vaccine candidates amongst the most abundant constituents. The proteomic approach has been successfully applied to identify the secretions used by cercaria to penetrate host skin, the gut secretions of adult worms and the proteins exposed on the tegument surface. Soluble proteins can also be separated by 2D electrophoresis before western blotting to identify the full range of antigenic targets present in a parasite preparation. The next step is to discover which target proteins represent the weak points in the worm's defences.

Data set of Aspergillus flavus induced alterations in tear proteome: Understanding the pathogen-induced host response to fungal infection

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Jeyalakshmi Kandhavelu

2016-12-01

Full Text Available Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled “Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection [1]” (Jeyalakhsmi Kandhavelu, Naveen Luke Demonte, Venkatesh Prajna Namperumalsamy, Lalitha Prajna, Chitra Thangavel, Jeya Maheshwari Jayapal, Dharmalingam Kuppamuthu, 2016. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.

The path to enlightenment: making sense of genomic and proteomic information.

Science.gov (United States)

Maurer, Martin H

2004-05-01

Whereas genomics describes the study of genome, mainly represented by its gene expression on the DNA or RNA level, the term proteomics denotes the study of the proteome, which is the protein complement encoded by the genome. In recent years, the number of proteomic experiments increased tremendously. While all fields of proteomics have made major technological advances, the biggest step was seen in bioinformatics. Biological information management relies on sequence and structure databases and powerful software tools to translate experimental results into meaningful biological hypotheses and answers. In this resource article, I provide a collection of databases and software available on the Internet that are useful to interpret genomic and proteomic data. The article is a toolbox for researchers who have genomic or proteomic datasets and need to put their findings into a biological context.

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

Science.gov (United States)

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment

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Sihlbom Carina

2011-09-01

Full Text Available Abstract Background Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions. Methods Endobronchial biopsies were taken from untreated asthmatic patients (n = 12 and healthy controls (n = 3. Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment. Results More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo. Conclusions Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

Science.gov (United States)

Park, Gun Wook; Hwang, Heeyoun; Kim, Kwang Hoe; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Ji Yeong; Ji, Eun Sun; Park, Sung-Kyu Robin; Yates, John R; Kwon, Kyung-Hoon; Park, Young Mok; Lee, Hyoung-Joo; Paik, Young-Ki; Kim, Jin Young; Yoo, Jong Shin

2016-11-04

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

"Does understanding the brain need proteomics and does understanding proteomics need brains?"--Second HUPO HBPP Workshop hosted in Paris.

Science.gov (United States)

Hamacher, Michael; Klose, Joachim; Rossier, Jean; Marcus, Katrin; Meyer, Helmut E

2004-07-01

The second Human Brain Proteome Project (HBPP) Workshop of the Human Proteome Organisation (HUPO) took place at the Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI) from April 23-24, 2004. During two days, more than 70 attendees from Europe, Asia and the US came together to decide basic strategic approaches, standards and the beginning of a pilot phase prior to further studies of the human brain proteome. The international consortium presented the technological and scientific portfolio and scheduled the time table for the next year.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

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Marlena M Wilson

Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis.

Science.gov (United States)

Yu, Yadong; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

2017-03-07

Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.

Regional differences of the urinary proteomes in healthy Chinese individuals

OpenAIRE

Qin, Weiwei; Wu, Jianqiang; Pan, Li; Zhang, Fanshuang; Wang, Xiaorong; Zhang, Biao; Shan, Guangliang; Gao, Youhe

2017-01-01

Urine is a promising biomarker source for clinical proteomics studies. Although regional physiological differences are common in multi-center clinical studies, the presence of significant differences in the urinary proteomes of individuals from different regions remains unknown. In this study, morning urine samples were collected from healthy urban residents in three regions of China and urinary proteins were preserved using a membrane-based method (Urimem). The urine proteomes of 27 normal s...

Global phosphotyrosine proteomics identifies PKCδ as a marker of responsiveness to Src inhibition in colorectal cancer.

Directory of Open Access Journals (Sweden)

Eliot T McKinley

Full Text Available Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC. We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ, CUB-domain-containing protein 1 (CDCP1, Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2, and receptor protein-tyrosine phosphatase alpha (RPTPα. The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.

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....

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.