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beruecksichtige naturwissenschaftliche Journale:
Molecular & Cellular Proteomics - published by
Society for Biochemistry and Molecular Biology -
... will publish three types of original articles: research papers, database articles and technology development articles.
Journal of Proteome Research - published by
The American Chemical Society -
... integrates the fields of chemistry, mathematics, applied physics, biology, and medicine in order to better understand the function of proteins in biological systems.
Proteomics - published by
Wiley-Interscience -
... is the premier international source for information on all aspects of applications and technologies in proteomics. It provides the most comprehensive coverage in the field, spanning significant technical developments in all major gel and non-gel based proteomic platforms and their application in every area of life sciences.
Aktuelle wissenschaftliche Fachartikel der
genannten Journale:
The CA 19-9 antigen is currently the best individual marker for the detection of pancreatic cancer. In order to optimize the CA 19-9 assay and to develop approaches to further improve cancer detection, it is important to understand the specificity differences between CA 19-9 antibodies and the consequential affect on biomarker performance. Antibody arrays enabled multiplexed comparisons between five different CA 19-9 antibodies used in the analysis of plasma samples from pancreatic cancer patients and controls. Major differences were observed between antibodies in their detection of particular patient samples. Glycan array analysis revealed that certain antibodies were highly specific for the canonical CA 19-9 epitope, sialyl-Lewis A, while others bound sialyl-Lewis A in addition to a related structure called sialyl-Lewis C and modification with Nue5Gc. In a much larger patient cohort we confirmed the binding of Sialyl-Lewis C glycan by one of the antibodies and showed that the broader specificity led to the detection of an increased number of cancer patients without increasing detection of pancreatitis patient samples. This work demonstrates that variation between antibody specificity for cancer-associated glycans can have significant implications for biomarker performance and highlights the value of characterizing and detecting the range of glycan structures that are elevated in cancer.
ABL tyrosine kinase inhibitor (TKI) therapy induces clinical remission in chronic myeloid leukemia (CML) patients but early relapses and later emergence of TKI-resistant disease remain problematic. We recently demonstrated that the AHI-1 oncogene physically interacts with BCR-ABL and JAK2 and mediates cellular resistance to TKI in CML stem/progenitor cells. We now show that deletion of the SH3 domain of AHI-1 significantly enhances apoptotic response of BCR-ABL+ cells to TKIs compared to cells expressing full-length AHI-1. We have also discovered a novel interaction between AHI-1 and Dynamin-2, a GTPase, through the AHI-1 SH3 domain. The crystal structure of the AHI-1 SH3 domain at 1.53-Å resolution reveals that it adopts canonical SH3 folding, with the exception of an unusual C-terminal α helix. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between the AHI-1 SH3 domain and its ligands. These studies showed that an “Arg-Arg-Trp” stack may form within the binding interface, providing a potential target site for designing specific drugs. The crystal structure of the AHI-1 SH3 domain thus provides a valuable tool for identification of key interaction sites in regulation of drug resistance and for the development of small molecule inhibitors for CML.
Ceramide is a bioactive molecule involved in numerous cell signaling pathways that are associated with cell cycle control, differentiation, senescence and apoptosis. Although substantial knowledge about ceramide-regulated pathways has accumulated in the past decade, molecular mechanisms of ceramide action remain poorly understood, primarily due to limited information about ceramide-binding proteins. In the present study, we used affinity purification with a synthetic biotin-conjugated C6-ceramide analogue and LC-MS/MS to identify potential ceramide-interacting proteins in D6P2T Schwannoma cells. The purification resulted in identification of 97 unique proteins. The identified proteins are involved in various cellular processes, including apoptosis, cellular stress, cell cycle, cell differentiation, signaling, transcription, translation, protein biogenesis, metabolism and transport.
In this review we present an update on the progress of the Human Protein Atlas, with an emphasis on strategies for validating immunohistochemistry-based protein expression patterns and on the possibilities to extend the map of protein expression patterns for cancer research projects. The objectives underlying the Human Protein Atlas include i) the generation of validated antibodies towards a major isoform of all proteins encoded by the human genome, ii) creating an information database of protein expression patterns in normal human tissues, in cells and in cancer, and iii) utilizing generated antibodies and protein expression data as tools to identify clinically useful biomarkers. The success of such an effort is dependent on the validity of antibodies as specific binders of intended targets in applications used to map protein expression patterns. The development of strategies to support specific target binding is crucial and remains a challenge as a large fraction of proteins encoded by the human genome is poorly characterized, including the approximately 1/3 of all proteins lacking evidence of existence. Conceivable methods for validation include the use of paired antibodies, i.e. two independent antibodies targeting different and non-overlapping epitopes on the same protein as well as comparative analysis of mRNA expression patterns with corresponding proteins.
Acute myeloid leukemia (AML) patients present with cancerous cells originating from bone marrow. Proteomic data on AML patient cells provides critical information on the key molecules associated with the disease. Here we introduce a new computational approach to identify complex patterns in protein signaling from reverse phase protein array data. We analyzed the expression of 203 proteins in cells taken from AML patients. Dominant overlapping protein networks between subtypes of AML patients were characterized computationally, through a paired t-test approach looking at relative protein expression. In the first application of this method, we compared recurrent cytogenetic abnormalities inv(16) and t(8;21), both affecting core-binding factor CBFβ, to normal CD34+ cells and to each other. 678 sets of proteins were identified as significantly different in both inv(16) and t(8;21) compared to controls, at the Bonferroni number, α < 2.44 * 10−6. We strengthened our predictions by comparing results to those obtained using lasso regression analysis. Signaling networks were constructed from the protein pairs that were significantly different in the t-test and lasso regression analysis. Predicted networks were also compared to known networks from public protein-protein interaction and signaling databases. By characterizing unique “protein signatures” through this rapid computational analysis, and placing them in the context of canonical biological networks, we identify signaling pathways distinct to subcategories of AML patients.
Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signalling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0) and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signalling processes. Furthermore, we demonstrate colocalisation of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor–specific signalling events.
Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D-nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser 431 and Ser 432, and ubiquitination site located at Lys 444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.
Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery. First, primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients, and the serum-free conditioned medium (CM) samples were collected for proteomic analysis. The total proteins from the CM sample were separated by SDS-PAGE, digested with trypsin and then analyzed by LC-MS/MS. Combining data from the tumor tissues and the normal tissues, 1129 proteins were identified in total, of which those categorized as ‘extracellular proteins’ and ‘plasma membrane proteins’ accounted for 21.4% and 16.9%, respectively. For validation, three secretory proteins (NID1, TIMP2 and VCAN) that involved in ‘organ development’-associated sub-network, showed significant differences between their levels in the circulating plasma samples from ovarian cancer patients and healthy women. In conclusion, this ovarian cancer-derived protein database provides a credible repertoire of potential biomarkers in blood for this malignant disease, and deserves mining further.
Post-translational modifications of extracellular domains of membrane proteins can influence antibody binding and give rise to ambivalent results. Best prove of protein expression are the use of complementary methods to provide unequivocal evidence. CXCR7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL12 and CXCL11. The expression of CXCR7 on non-hematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was recently challenged. To solve the dissent we thoroughly analyzed the expression of CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. Examination of CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human B cells. Moreover, real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally two CXCR7-specific peptides were identified by mass spectrometry in immunoprecipitates from primary human B cells. Thus, we present a strategy, based on combined proteomic and functional approaches, that can be used to solve dissents on protein expression, i.e. demonstrating the expression of CXCR7 on human leukocytes.
LTQ Orbitrap data analyzed with ProteinPilot can be further improved by MaxQuant raw data processing, which utilizes precursor-level high mass accuracy data for peak processing and MGF creation. In particular, ProteinPilot results from MaxQuant-processed peaklists for Orbitrap datasets resulted in improved spectral utilization due to an improved peaklist quality with higher precision and precursor mass accuracy (HPMA). The output and post-search analysis tools of both workflows were utilized for previously unexplored features of a 3D-fractionated and hexapeptide library (ProteoMiner) treated whole saliva dataset comprising 200 fractions. ProteinPilot's ability to simultaneously predict multiple modifications showed an advantage from ProteoMiner treatment for modified peptide identification. We demonstrate that complementary approaches in the analysis pipeline provide comprehensive results for the whole saliva dataset acquired on an LTQ Orbitrap. Overall our results establish a workflow for improved protein identification from high mass accuracy data.
The surface proteome (surfaceome) of the marine planctomycete Rhodopirellula baltica SH1T was studied using a biotinylation and a proteinase K approach combined with SDS-PAGE and mass spectrometry 52 of the proteins identified in both approaches could be assigned to the group of potential surface proteins. Among them are some high molecular weight proteins, potentially involved in cell-cell attachment, that contain domains shown before to be typical for surface proteins like cadherin/ dockerin domains, a bacterial adhesion domain or the fasciclin domain. The identification of proteins with enzymatic functions in the R. baltica surfaceome provides further clues for the suggestion that some degradative enzymes may be anchored onto the cell surface. YTV proteins, which have been earlier supposed to be components of the proteinaceous cell wall of R. baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin /N-methylation domain and a planctomycete-typical DUF1559 domain were identified.
The virulence of influenza virus is determined by viral and host factors. Data on the genetic basis of the virulence of H5N1 influenza viruses have increased over the past decade;, however, the contributions of host factors to the outcomes of H5N1 infection remain largely unknown. Here, we tested two chicken H5N1 viruses in mice and found that A/chicken/VN1214/2007 was non-lethal in mice and only replicated in the lung, whereas A/chicken/VN1180/2006 was highly lethal and replicated systemically in mice. To investigate the host response against these two different virus infections, we performed proteomic analysis by using 2-D Fluorescence Difference Gel Electrophoresis on the lung tissues of mice collected on day 1 and day 3 post-inoculation with different viruses or PBS as a control. Thirty-nine differentially expressed (DE) proteins related to “immune and stimulus response”, “macromolecular biosynthesis and metabolism”, and “cellular component and cytoskeleton” were identified in the virus-inoculated groups. Moreover, 13 DE proteins were identified between the two virus-inoculated groups, implying that these proteins may play important roles in the different outcomes of infection with these two viruses. Our data provide important information regarding the host response to mild and lethal H5N1 influenza virus infection.
Oncovirus, synonymously called a “tumour virus” is a virus that can cause cancer. An oncolytic virus preferentially infects the host's cancer cells and lyses them, causing tumour destruction, and is thus referred to as a “cancer killing virus”. With an estimated 11% of cancer associated deaths caused by oncoviruses and the possibility that many cancers may be treated by using oncolytic viruses, the role of viruses in cancer may be viewed as a double edged sword. A total of seven human cancer viruses have been identified as oncoviruses, having been associated with various cancers. Conversely, a large number of oncolytic viruses have shown great potential towards the treatment of certain types of cancer. Proteomics has now been applied towards understanding the complex interplay that exists between oncoviruses and the immune responses that serve to prevent oncoviral diseases. This review attempts to summarise the neoplastic potential of human tumour associated viruses and associated vaccine successes. The potential use of oncolytic viruses for the therapeutic intervention of cancer will also be discussed. Finally, this review will discuss the enormous potential of proteomics technology in the field of oncovirology.
Proteomic analysis on sperm has been restricted to only a few model organisms. We present here a 2-DE PAGE proteome map of sperm cells from a non-model organism, the marine mussel Mytilus edulis, a free-spawning marine invertebrate with external fertilization. Ninety-Six protein spots showing high expression were selected and of these 77 were successfully identified by nESI-MS analysis. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results and proteomics approach used are discussed in relation to their potential for advancing understanding of the unusual system of mtDNA inheritance described in Mytilus spp, and for the testing of evolutionary hypotheses pertaining to the role of fertilisation in the speciation process.
Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2-DE and mass spectrometry to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in “signal transduction” and “lipid metabolism”, relative expression levels of proteins containing signal peptides, and proteins in “defense and stress response”. These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level.
To better understand the involvement of Retinal Müller glial cells (RMG) in retinal diseases, we phenotyped primary porcine RMG in dependence of cultivation time using different quantitative proteomic strategies. A well-established LC-MS/MS-based quantification method was employed: stable isotope labeling by amino acids in cell culture (SILAC) and directly compared to label-free (LF) quantifications, based on total peak intensities using two different programs (MaxQuant and Progenesis LC-MS). The overall numbers of detected proteins were largely similar (overlap of 1324 proteins), only a total of 173 proteins were significantly altered between the different culture conditions. However, among these, only 21 proteins were shared between the three analytical strategies. Hence, the majority of altered proteins only reached significance thresholds in one of the applied analyses with a larger overlap between the two LF approaches. Among the shared differentially abundant proteins were known RMG markers as well as new proteins associated with glial cell transition. However, proteins correlated to cellular transitions and dedifferentiation were also found among the proteins only significant in one or two of the applied strategies. Consequently, the application of different quantification and analytical strategies could increase the analytical depths of proteomic phenotyping.
Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and challenge in proteolytically modified protein study to identify physiologic protease–substrate relationships and find out potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L and HCCLM6, were researched with a high throughput and sensitive PROTOMAP platform. Total 391 proteins were found proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatics tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered sub-network, including SPTBN1, ATP5B and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role on human HCC metastasis.
S-nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O6-alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR−/−) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR−/− mice compared to those in the wild-type mice. We subsequently identified 19 up-regulated and 19 down-regulated proteins in GSNOR−/− mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR−/− mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR−/− mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.
Secreted proteins play a key role in cell signalling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of “death ligands”. Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signalling. Differential proteomic analysis of conditioned media using 2-dimensional gel electrophoresis resulted in detection of numerous spots which were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI TOF MS and LC-MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in iso-electric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by western-blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post-translational modifications, including an alternating removal of N-terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of inter-cells relationships and radiosensitivity.
Benzo(a)pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal–processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage only few data has been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.
The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water soluble proteins were separated into HMW, MMW and LMW fractions by size-exclusion HPLC and constituents of each protein class revealed by 1-DE and 2-DE. Spots were selected from 2-DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre there was a decrease in α and β-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin / quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins and their function.
The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins was identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (non-muscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically-validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors vs. corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.
Chronic alcohol consumption culminates in alcoholic hepatitis which is characterized by ballooning degeneration of hepatocytes and perivenous inflammation. The aldehydes produced by ethanol oxidation and lipid peroxidation form adducts with the hepatic proteins rendering them immunogenic and initiating an autoimmune response. The present study was designed to identify these immunoreactive hepatic proteins in ethanol treated Balb/c mice. Liver cytosolic, mitochondrial and microsomal proteins from the ethanol treated and control female Balb/c mice were size fractionated on SDS-PAGE and immunoblotted with the sera from the individual animal. The immunoreactive proteins were identified using anti- mouse IgG antibody and characterized by MALDI-TOF. It is the first report demonstrating that 15 hepatic proteins show immunoreactivity following alcohol administration. The identified autoreactive proteins ranged in function from metabolism to cytoskeletal support. Remarkably, 3 key enzymes of ethanol metabolism namely ADH, ALDH I and III as well as important antioxidant enzyme GST were found to be autoreactive upon ethanol treatment. We conclude that ethanol treatment induces biotransformation of host proteins from almost every compartment of the cell especially the enzymes involved in the detoxification of ethanolic insult being the major target for biotransformation. Hence we propose that these proteins can be the potential candidates for the biomarker studies.
Proteomic analyses of cartilage and, to a lesser extent, of bone have long been impaired because of technical challenges related to their structure and biochemical properties. We have developed a unified method based on phenol extraction, 2-DE, silver staining and subsequent LC-MS/MS. This method proved to be efficient to characterize the proteome of equine cartilage and bone samples collected in vivo. Since proteins from several cellular compartments could be recovered, our procedure is mainly suitable for in situ molecular physiology studies focused on the cellular content of chondrocytes, osteoblasts and osteoclasts as well as that of the extracellular matrix (ECM), with the exception of proteoglycans. Our method alleviates some drawbacks of cell culture that can mask physiological differences, as well as reduced reproducibility due to fractionation. Proteomic comparative studies between cartilage and bone samples from healthy and affected animals were thus achieved successfully. This achievement will contribute to increasing knowledge on the molecular mechanisms underlying the physiopathology of numerous osteoarticular diseases in horses and in humans.
In this study, we presented a quality control tool named PepDistiller to facilitate the validation of MASCOT search results. By including the number of tryptic termini, and integrating a refined false discovery rate (FDR) calculation method, we demonstrated the improved sensitivity of peptide identifications obtained from semi-tryptic search results. Based on the analysis of a complex dataset, approximately 7% more peptide identifications were obtained using PepDistiller than using MASCOT Percolator. Moreover, the refined method generated lower FDR estimations than the percentage of incorrect target (PIT)-fixed method applied in Percolator. Using a standard dataset, we further demonstrated the increased accuracy of the refined FDR estimations relative to the PIT-fixed FDR estimations. PepDistiller is fast and convenient to use, and is freely available for academic access. The software can be downloaded from http://bioinfo.hupo.org.cn/tools/PepDistiller.
The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on Saccharomyces cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild type BY4741 strain grown on fermentable and on non-fermentable carbon sources. The analysis showed that on non-fermentable medium the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol this mutant displayed an increase of some glycolytic and fermentative enzymes such as Glyceraldehyde-3-phosphate dehydrogenase 1, Enolase 2, Pyruvate decarboxylase 1 and Alchol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on non-fermentable carbon source the lack of Sco1p causes a metabolic shift, from respiration to fermentation.
Viruses induce dramatic changes in target tissue during pathogenesis, including host cellular responses that either limit or support the pathogen. The infectious bursal disease virus (IBDV) targets primarily the bursa of Fabricius (BF) of chickens, causing severe immunodeficiency. Here we characterized the cellular proteome changes of the BF caused by IBDV replication in vivo using two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS) identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared between 24 and 96 h after IBDV infection. Mass spectrometry identified 54 altered cell proteins, 12 of which were notably up-regulated by IBDV infection. Meanwhile, the other 42 cellular proteins were considerably suppressed by IBDV infection and are involved in protein degradation, energy metabolism, stress response, host macromolecular biosynthesis and transport process. The up-regulation of β-actin and down-regulation of dynamin during IBDV infection were also confirmed by Western blot and immunofluorescence analysis. These altered protein expressions provide a response profile of chicken BF to virulent IBDV infection. Further functional study on these altered proteins may lead to better understanding of pathogenic mechanisms of virulent IBDV infection and to new potential therapeutic targets.
Shewanella inhabit a wide variety of niches in nature and can utilize a broad spectrum of electron acceptors under anaerobic conditions. How they modulate their gene expression to adapt is poorly understood. ArcA, homologue of a global regulator controlling hundreds of genes involved in aerobic and anaerobic respiration in E. coli, was shown to be important in aerobiosis/anaerobiosis of S. oneidensis as well. Loss of ArcA, in addition to altering transcription of many genes, resulted in impaired growth under aerobic condition, which was not observed in E. coli. To further characterize the impact of ArcA loss on gene expression on the level of proteome under aerobic and anaerobic conditions, liquid-chromatography-mass-spectrometry (LC-MS) based proteomic approach was employed. Results show that ArcA loss led to globally altered gene expression, generally consistent with that observed with transcripts. Comparison of transcriptomic and proteomic data permitted identification of 17 high-confidence ArcA targets. Moreover, our data indicate that ArcA is required for regulation of cytochrome c proteins, and the menaquinone level may play a role in regulating ArcA as in E. coli. Proteomic-data-guided growth assay revealed that the aerobic growth defect of ArcA mutant is presumably due to impaired peptide utilization.
Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.
Posterior cranial fossa is the most frequent location of paediatric brain tumours. Its diagnosis is currently performed by post-surgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less-invasive tool.
Patient CSF was collected during surgery before the tumour removal (PRE-CSF) and six days after the resection (POST-CSF) and analysed by top-down LC-MS proteomics for comparison. The PRE-CSFs generally exhibited a less complex LC-MS profile than the relative POST-CSFs suggesting a suppressive role of the tumour towards proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha- and beta- hemoglobin chains fragments, resulted generally absent in the PRE-CSF and present in the POST- ones independently from contaminant blood hemoglobin. Among them, the LVV- and VV-hemorphin-7 showed the most repeatable trend and a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to presence of tumour in the patient despite surgery due to metastases or to sub-total resection.
These results ascribed a relevant biological role to LVV- and VV-h7 peptides inside the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used in supporting the prediction of a good patient prognosis.
Drosophila melanogaster has been used as a genetic model organism to understand the fundamental molecular mechanisms in human biology including memory formation which has been reported involving protein synthesis and/or post-translational modification. In this study, we employed a proteomic platform based on fluorescent two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to build a standard Drosophila 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 Drosophila 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.
Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by isoelectric point to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (∼2 week), inexpensive and scalable purification of arg/lys labeled IQcat standards in the E. coli auxotroph AT713. With this pipeline we assess the fidelity of IQcat-based absolute quantification for 10 yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.
The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1–D gel electrophoresis, reversed–phase liquid chromatography, and tandem mass spectrometry to analyze the excretion–secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion–secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.
To improve the understanding of microbial behaviors in communities, proteomic tracking, an approach for relative quantification of species-specific population dynamics of mixed cultures, was developed. Therefore, a bacterial mixed culture was analyzed during batch cultivations with and without addition of the antibiotic Ceftazidime. The community was composed of Burkholderia cepacia, Pseudomonas aeruginosa, and Staphylococcus aureus, pathogens causing infections in cystic fibrosis patients. Gel-based proteomics and mass-spectrometry were used to obtain qualitative and quantitative proteomic data. During cultivation P. aeruginosa became dominant within the mixed culture while S. aureus was inhibited in growth. Analysis of samples – taken along cultivation – revealed about 270 differentially expressed proteins. Some of those proteins related to bacterial interactions, response to antibiotic treatment, and metabolic shifts. For instance, the enzymes PhzS, PhzD and PhzG2 indicated the production of the antibiotic pigment pyocyanine by P. aeruginosa which is related to oxidative stress and therefore, might inhibit growth of S. aureus. Overall, the strategy applied not only allows species-specific tracking of the community composition but also provides valuable insights into the behavior of mixed cultures.
Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H2O2-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H2O2 resistance of exponential growing cells. Upon H2O2 exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be up-regulated and disruption of arcA or arcB genes abolished H2O2 resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.
The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulation of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed up-regulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and up-regulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.
The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included Superoxide dismutase 1 (SOD1), Apolipoprotein A-I (ApoA-I), and Dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1 and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.
The development of ion mobility (IM) mass spectrometry instruments has the capability to provide an added dimension to peptide analysis pipelines in proteomics, but, as yet, there are few software tools available for analysing such data. IM can be used to provide additional separation of parent ions or product ions following fragmentation. In this work, we have created a set of software tools that are capable of converting three dimensional IM data generated from analysis of fragment ions into a variety of formats used in proteomics. We demonstrate that IM can be used to calculate the charge state of a fragment ion, demonstrating the potential to improve peptide identification by excluding non-informative ions from a database search. We also provide preliminary evidence of structural differences between b and y ions for certain peptide sequences but not others. All software tools and data sets are made available in the public domain at http://code.google.com/p/ion-mobility-ms-tools/.
A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrial barrel-like structures of four-stacked homoheptameric rings of α- and β-type subunits organized in α7β7β7α7 stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α17β7β7α17 and α27β7β7α27 CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry (CXMS), together with the available structural data of non-halophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and β-homoheptameric rings from among several different possible PDB structures.
A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with two-dimensional gels and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on brain creatine kinase (Ckb) and triose phosphate isomerase 1 (Tpi1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.
Docetaxel is a taxane-derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone-refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multifactoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy.
To identify proteins altered in the resistant phenotype three parental cell lines DU145, 22RV1 and PC-3, as well as their docetaxel resistant sub-lines, were subjected to quantitative label-free LC-MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub-lines, 254 in the 22RV1 sub-lines, and 51 and 72 in the 8 and 12 nM resistant PC-3 sub-lines respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across 2 or more resistant variants. These included proteins indicative of an epithelial to mesenchemyl transition as well as altered heat shock response elements.
A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC50 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increasedwith treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells.
Modular protein interaction domains (PIDs) that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some PIDs such as SH2, 14–3-3, Chromo, and Bromo domains serve to recognize posttranslational modification (PTM) of amino acids (such as phosphorylation, acetylation, methylation, etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PSD-95/Discs-large/ZO-1 (PDZ) domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate-specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High-throughput analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry, or other HTP techniques provides new insight into the potential protein–protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein–protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls of high-throughput analysis of protein–peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of PIDs and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions.
Our ability to study protein-protein interactions has grown by leaps and bounds in recent years, enabling numerous large-scale studies to be performed in a variety of organisms. Despite this success, some classes of proteins, including those bound to chromatin, remain difficult to characterize through proteomic approaches. Some of the problems faced by researchers studying chromatin-bound proteins include low complex solubility, heterogeneous sample composition, and numerous transient interactions which can be further complicated by the presence of DNA itself. To tackle these issues, a number of innovative protocols have been developed to better study the various facets of chromatin biology. In this review, we will discuss novel approaches to study protein-DNA interactions as well as protein complexes affecting chromatin.
Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area.
Analysis of protein interaction networks and protein complexes using affinity purification and mass spectrometry (AP/MS) is among most commonly used and successful applications of proteomics technologies. One of the foremost challenges of AP/MS data is a large number of false positive protein interactions present in unfiltered datasets. Here we review computational and informatics strategies for detecting specific protein interaction partners in AP/MS experiments, with a focus on incomplete (as opposite to genome-wide) interactome mapping studies. These strategies range from standard statistical approaches, to empirical scoring schemes optimized for a particular type of data, to advanced computational frameworks. The common denominator among these methods is the use of label-free quantitative information such as spectral counts or integrated peptide intensities that can be extracted from AP/MS data. We also discuss related issues such as combining multiple biological or technical replicates, and dealing with data generated using different tagging strategies. Computational approaches for benchmarking of scoring methods are discussed, and the need for generation of reference AP/MS datasets is highlighted. Finally, we discuss the possibility of more extended modeling of experimental AP/MS data, including integration with external information such as protein interaction predictions based on functional genomics data.
Identifying the interactions established by a protein of interest can be a critical step in understanding its function. This is especially true when an unknown protein of interest is demonstrated to physically interact with proteins of known function. While many techniques have been developed to characterize protein-protein interactions, one strategy that has gained considerable momentum over the past decade for identification and quantification of protein-protein interactions, is affinity purification followed by mass spectrometry (AP-MS). Here, we briefly review the basic principles used in affinity purification coupled to mass spectrometry, with an emphasis on tools (both biochemical and computational), which enable the discovery and reporting of high quality protein-protein interactions.
Molecular interaction databases are playing an ever more important role in our understanding of the biology of the cell. An increasing number of resources exist to provide these data and many of these have adopted the controlled vocabularies and agreed-upon standardized data formats produced by the Molecular Interaction workgroup of the Human Proteome Organization Proteomics Standards Initiative (HUPO PSI-MI). Use of these standards allows each resource to establish PSICQUIC service, making data from multiple resources available to the user in response to a single query. This cooperation between databases has been taken a stage further, with the establishment of the IMEx consortium which aims to maximize the curation power of numerous data resources, and provide the user with a non-redundant, consistently annotated set of interaction data.
Membrane-bound proteins are one of the most important protein types in the cell, and are involved in many major cell processes and signaling pathways. Most proteins, including those at membranes, must interact with other proteins to form complexes which are essential for their function. In this review, we describe some of the major non-mass spectrometry-based methods and technologies used for the investigation of intracellular membrane protein complexes, including Tango, fluorescence/bioluminescence resonance energy transfer (F/BRET), luminescence-based mammalian interactome mapping (LUMIER), protein-fragment complementation assay (PCA), and membrane yeast two-hybrid assay (MYTH). We highlight the advantages and drawbacks of these methods, describe recent studies utilizing these methods, and discuss some of the major findings in the study of membrane protein-based cell pathways.
Network visualization of the interactome has been become routine in systems biology research. Not only does it serve as an illustration on the cellular organization of protein-protein interactions, it also serves as a biological context for gaining insights from high-throughput data. However, the challenges to produce an effective visualization have been great owing to the fact that the scale, biological context, and dynamics of any given interactome are too large and complex to be captured by a single visualization. Visualization design therefore requires a pragmatic tradeoff between capturing biological concept and being comprehensible. In this review, we focus on the biological interpretation of different network visualizations. We will draw on examples predominantly from our experiences but elaborate them in the context of the broader field. A rich variety of networks will be introduced including interactomes and the complexome in 2D, interactomes in 2.5D and 3D, and dynamic networks.
Label-based quantitative mass spectrometry analysis of affinity purified complexes, with its built-in negative controls and relative ease of use, is an increasingly popular choice for defining protein-protein interactions and multiprotein complexes. This approach, which differentially labels proteins/peptides from two or more populations and combines them prior to analysis, permits direct comparison of a protein pulldown (e.g. affinity purified tagged protein) to that of a control pulldown (e.g. affinity purified tag alone) in a single mass spectrometry (MS) run, thus avoiding the variability inherent in separate runs. The use of quantitative techniques has been driven in large part by significant improvements in the resolution and sensitivity of high-end mass spectrometers. Importantly, the availability of commercial reagents and open source identification/quantification software has made these powerful techniques accessible to non-specialists. Benefits and drawbacks of the most popular labeling-based approaches are discussed here, and key steps/strategies for the use of labeling in quantitative immunoprecipitation experiments detailed.
Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many proteins and complexes as they exist in cells, tissues or other complex samples. This review will describe rationale, challenges and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies.
The physical interaction of proteins is subject to intense investigation that has revealed that proteins are assembled into large densely connected networks. In this review, we will examine how signaling pathways can be combined to form higher order protein interaction networks. By using network graph theory, these interaction networks can be further analyzed for global organization, which has revealed unique aspects of the relationships between protein networks and complex biological phenotypes. Moreover, several studies have shown that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer progression. These relationships suggest a novel paradigm for treatment of complex multigenic disease where the protein interaction network is the target of therapy more so than individual molecules within the network.
Protein interactions have been at the focus of computational biology in recent years. In particular, interest has come from two different communities—structural and systems biology. Here, we will discuss key systems and structural biology methods that have been used for analysis and prediction of protein–protein interactions and the insight these approaches have provided on the nature and organization of protein–protein interactions inside cells.
Cellular functions are defined by the dynamic interactions of proteins within macromolecular networks. Deciphering these complex interplays is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems, from a simple bacterial cell to highly regulated neuronal cells or cancerous tissue. In the last decade, affinity purification (AP) coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their macromolecular assemblies. This review discusses recent advances in AP approaches, from cell lysis to the importance of sample preparation and the choice of AP matrix as well as the development of different epitope tags and strategies to study dynamic interactions, with an emphasis on RNA–protein interaction networks.
Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.
Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as “base-less”) are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/MSanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MSschemes. In the present experimental outcome, additional LC-MS/MSanalysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MSbased identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.
Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex-I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARγ co-activator 1α (PGC-1α) protein. Chronic oxidative stress suppresses the gene expression of PGC-1α but concomitant inhibition of the ubiquitin–proteasome system (UPS) can stabilize this co-activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.
New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444–2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease.
The betaproteobacterium “Aromatoleum aromaticum” EbN1 utilizes eight different plant-derived nonhydroxylated (e.g. cinnamate) and hydroxylated (e.g. p-coumarate) 3-phenylpropanoids with nitrate as electron acceptor. Differential protein profiling (2D-DIGE) revealed abundance increases of five proteins (EbA5316 to EbA5320) during anaerobic growth with cinnamate, hydrocinnamate, p-coumarate, and 3-(4-hydroxyphenyl)propanoate, compared to anaerobic benzoate-adapted cells serving as reference state. The predicted functions of four of these proteins (EbA5317, fatty acid-coenzyme A (CoA) ligase; EbA5318, enoyl-CoA hydratase/isomerase; EbA5319, β-ketothiolase; and EbA5320, 3-hydroxyacyl-CoA dehydrogenase) suggest β-oxidation of the above 3-phenylpropanoids to benzoyl-CoA and p-hydroxybenzoyl-CoA, respectively. The fifth protein (EbA5316, ABC-type periplasmic solute-binding protein) could be involved in 3-phenylpropanoid uptake. The detection of 3-hydroxy-3-phenylpropanoate during anaerobic growth with cinnamate and hydrocinnamate or 3-hydroxy-3-(4-hydroxyphenyl)propanoate during anaerobic growth with p-coumarate and 3-(4-hydroxyphenyl)propanoate supports the proteome-predicted β-oxidation pathway. Based on the specific formation of EbA5316–20 also during anaerobic growth with further 3-phenylpropanoid growth substrates including cinnamyl alcohol, m-coumarate, 3-(3,4-dihydroxyphenyl)propanoate and 3,4-dihydroxycinnamate (caffeate), a common β-oxidation route is proposed for 3-phenylpropanoid degradation in strain EbN1. The low amount of metabolites attributable to cometabolic transformation of nongrowth supporting 3-phenylpropanoids (e.g. o-coumarate, ferulate) may be indicative for a high substrate specificity of the involved enzymes.
Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.
β-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.
Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MODi and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = −34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of −34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO2-SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.
Hepatitis B can progress into hepatocellular carcinoma. Body irons may interfere with the clearance of hepatitis B virus (HBV) and contribute to genesis of tumor. To investigate the role of iron played in HBV-related pathogenesis, here we studied the effect of iron with different concentrations and valence states on growth of HepG2.2.15 cells and secretion of virus proteins. A strong tolerance of HepG2.2.15 cells to iron challenge was found. The concentration of hepatitis B surface antigen in cell culture medium was decreased after iron stimulation. Lower concentrations of iron facilitated hepatitis B e-antigen (HBeAg) secretion. Fe2+ appeared more effective on HBeAg secretion than Fe3+ did. In parallel, the differential protein profiles in HepG2.2.15 cells were studied by iTRAQ and LC-MS/MS. The differentially expressed proteins were mainly involved in stress response, signal transduction, apoptosis, etc. Four proteins (14-3-3 β/α, VCP, migration inhibitory factor, and Nup153) were verified by Western-blotting and found to be consistent with the iTRAQ data. Interestingly, nuclear import of Nuclear factor kappa B (NFκB) and its activity were found to be affected by the decreased Nup153 in iron stimulated HepG2.2.15 cells. The results may indicate possible molecular mechanism how the synergism of HBV and iron stimulation damages host liver cells.
Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0–G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.
For MALDI analysis of glycans and glycopeptides, the choice of matrix is crucial in minimizing desialylation by mass spectrometric in-source and metastable decay. Here, we evaluated the potential of 4-chloro-α-cyanocinnamic acid (Cl-CCA) for MALDI-TOF-MS analysis of labile sialylated tryptic N-glycopeptides and released N- and O-glycans. Similar to DHB, but in contrast to CHCA, the Cl-CCA matrix allowed the analysis of sialylated N-glycans and glycopeptides in negative ion mode MALDI-TOF-MS. Dried droplet preparations of Cl-CCA provided microcrystals with a homogeneous spatial distribution and high shot-to-shot repeatability similar to CHCA, which simplified the automatic measurement and improved the resolution and mass accuracy. Interestingly, reflectron-positive ion mode analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans with Cl-CCA revealed more complete profiles than with DHB and CHCA. In conclusion, we clearly demonstrate the high potential of this rationally designed matrix for glycomics and glycoproteomics.
We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilusHB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilusHB8.
Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing.
Rapidly increasing genomic data present new challenges for scientists: making sense of millions of amino acid sequences requires a systematic approach and information about their 3D structure, function, and evolution. Over the last decade, numerous studies demonstrated the fundamental importance of protein tandem repeats and their involvement in human diseases. Bioinformatics analysis of these regions requires special computer programs and databases, since the conventional approaches predominantly developed for globular domains have limited success. To perform a global comparative analysis of protein tandem repeats, we developed the Protein Tandem Repeat DataBase (PRDB). PRDB is a curated database that includes the protein tandem repeats found in sequence databanks by the T-REKS program. The database is available at http://bioinfo.montp.cnrs.fr/?r=repeatDB
We utilized a setup based on extensive pre-fractionation of proteolytic peptides and nanoflow reversed-phase LC-MS/MS to identify the (sub)proteome of human follicular fluid (FF). In this in-depth screen, 246 specific proteins were identified, the majority of which are involved in coagulation- and immune-response pathways. Our aim is to define a set of FF protein markers, which could predict oocyte quality.
Protein interactions mediate essentially all biological processes and analysis of protein–protein interactions using both large-scale and small-scale approaches has contributed fundamental insights to the understanding of biological systems. In recent years, interactome network maps have emerged as an important tool for analyzing and interpreting genetic data of complex phenotypes. Complementary experimental approaches to test for binary, direct interactions, and for membership in protein complexes are used to explore the interactome. The two approaches are not redundant but yield orthogonal perspectives onto the complex network of physical interactions by which proteins mediate biological processes. In recent years, several publications have demonstrated that interactions from high-throughput experiments can be equally reliable as the high quality subset of interactions identified in small-scale studies. Critical for this insight was the introduction of standardized experimental benchmarking of interaction and validation assays using reference sets. The data obtained in these benchmarking experiments have resulted in greater appreciation of the limitations and the complementary strengths of different assays. Moreover, benchmarking is a central element of a conceptual framework to estimate interactome sizes and thereby measure progress toward near complete network maps. These estimates have revealed that current large-scale data sets, although often of high quality, cover only a small fraction of a given interactome. Here, I review the findings of assay benchmarking and discuss implications for quality control, and for strategies toward obtaining a near-complete map of the interactome of an organism.
Ubiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K--GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K--GG peptides three- to fourfold resulting in detection of up to ~3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K--GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study.
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.
Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
The COP9 signalosome (CSN) is a multi-subunit protein complex that performs critical roles in controlling diverse cellular and developmental processes. Aberrant regulation of the CSN complex has been shown to lead to tumorigenesis. Despite its biological significance, our current knowledge of the function and regulation of the CSN complex is very limited. To explore CSN biology, we have developed and employed a new version of the tag team-based QTAX strategy (quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes) by incorporating a label-free MS method for quantitation. Coupled with protein interaction network analysis, this strategy produced a comprehensive and detailed assessment of the protein interaction network of the human CSN complex. In total, we quantitatively characterized 825 putative CSN-interacting proteins, with 270 classified as core interactors (captured by all three bait purifications). Biochemical validation further confirms the validity of selected identified interactors. This work presents the most complete analysis of the CSN interaction network to date, providing an inclusive set of physical interaction data consistent with physiological roles for the CSN. Moreover, the methodology described here is a general proteomic tool for the comprehensive study of protein interaction networks.
Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse.
Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon- signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.
Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells.
Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex.
Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism.
Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.
Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription.
Using enrichment strategies many research groups are routinely producing large data sets of post-translationally modified peptides for proteomic analysis using tandem mass spectrometry. Although search engines are relatively effective at identifying these peptides with a defined measure of reliability, their localization of site/s of modification is often arbitrary and unreliable. The field continues to be in need of a widely accepted metric for false localization rate that accurately describes the certainty of site localization in published data sets and allows for consistent measurement of differences in performance of emerging scoring algorithms. In this article are discussed the main strategies currently used by software for modification site localization and ways of assessing the performance of these different tools. Methods for representing ambiguity are reviewed and a discussion of how the approaches transfer to different data types and modifications is presented.
Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a ~50 MDa complex consisting of ~30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa must be aided across by transport factors, most of which belong to a related family of proteins termed karyopherins (Kaps). These transport factors bind to the disordered phenylalanine-glycine (FG) repeat domains in a family of NPC proteins termed FG nups, and this specific binding allows the transport factors to cross the NPC. However, we still know little in terms of the molecular and kinetic details regarding how this binding translates to selective passage of transport factors across the NPC. Here we show that the specific interactions between Kaps and FG nups are strongly modulated by the presence of a cellular milieu whose proteins appear to act as very weak competitors that nevertheless collectively can reduce Kap/FG nup affinities by several orders of magnitude. Without such modulation, the avidities between Kaps and FG nups measured in vitro are too tight to be compatible with the rapid transport kinetics observed in vivo. We modeled the multivalent interactions between the disordered repeat binding sites in the FG nups and multiple cognate binding sites on Kap, showing that they should indeed be sensitive to even weakly binding competitors; the introduction of such competition reduces the availability of these binding sites, dramatically lowering the avidity of their specific interactions and allowing rapid nuclear transport.
To understand how the chromosomal passenger complex ensures chromosomal stability, it is crucial to identify its substrates and to find ways to specifically inhibit the enzymatic core of the complex, Aurora B. We therefore developed a chemical genetic approach to selectively inhibit human Aurora B. By mutating the gatekeeper residue Leu-154 in the kinase active site, the ATP-binding pocket was enlarged, but kinase function was severely disrupted. A unique second site suppressor mutation was identified that rescued kinase activity in the Leu-154 mutant and allowed the accommodation of bulky N6-substituted adenine analogs. Using this analog-sensitive Aurora B kinase, we found that retention of the chromosomal passenger complex at the centromere depends on Aurora B kinase activity. Furthermore, analog-sensitive Aurora B was able to use bulky ATPS analogs and could thiophosphorylate multiple proteins in cell extracts. Utilizing an unbiased approach for kinase substrate mapping, we identified several novel substrates of Aurora B, including the nucleosomal-binding protein HMGN2. We confirmed that HMGN2 is a bona fide Aurora B substrate in vivo and show that its dynamic association to chromatin is controlled by Aurora B.
Correct classification of cancer patients into subtypes is a prerequisite for acute diagnosis and effective treatment. Currently this classification relies mainly on histological assessment, but gene expression analysis by microarrays has shown great promise. Here we show that high accuracy, quantitative proteomics can robustly segregate cancer subtypes directly at the level of expressed proteins. We investigated two histologically indistinguishable subtypes of diffuse large B-cell lymphoma (DLBCL): activated B-cell-like (ABC) and germinal-center B-cell-like (GCB) subtypes, by first developing a general lymphoma stable isotope labeling with amino acids in cell culture (SILAC) mix from heavy stable isotope-labeled cell lines. This super-SILAC mix was combined with cell lysates from five ABC-DLBCL and five GCB-DLBCL cell lines. Shotgun proteomic analysis on a linear ion trap Orbitrap mass spectrometer with high mass accuracy at the MS and MS/MS levels yielded a proteome of more than 7,500 identified proteins. High accuracy of quantification allowed robust separation of subtypes by principal component analysis. The main contributors to the classification included proteins known to be differentially expressed between the subtypes such as the transcription factors IRF4 and SPI1/PU.1, cell surface markers CD44 and CD27, as well as novel candidates. We extracted a signature of 55 proteins that segregated subtypes and contained proteins connected to functional differences between the ABC and GCB-DLBCL subtypes, including many NF-B-regulated genes. Shortening the analysis time to single-shot analysis combined with use of the new linear quadrupole Orbitrap analyzer (Q Exactive) also clearly differentiated between the subtypes. These results show that high resolution shotgun proteomics combined with super-SILAC-based quantification is a promising new technology for tumor characterization and classification.
Influenza A virus is one of the world's major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention
The 12th HLPP workshop was held in conjunction with the 9th HUPO 2010 World Congress on September 20th in Sydney, Australia. This 2-hour workshop was chaired by Prof. Fuchu He (Beijing Proteome Research Center, BPRC). Highlights included: 1) progress of the post-translational modification of liver proteome; 2) construction of the human liver protein interaction and localization maps; 3) progress made in terms of identifying biomarker for liver diseases; and 4) discussion on the second phase of the initiative. This was followed by a lively discussion related to the project.
Cerebellum is an important brain region involved in motor, cognition, learning and memory functions. Proteome mapping of the 21 days old rat cerebellum identified total 285 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 49 neuronal activity-specific proteins, 7 of which are reported for the first time from the cerebellum in this study. The protein sequence data for 31 proteins reported here has been integrated in the UniProt Knowledgebase.
The effective isolation and purification of proteins from biological fluids is the most crucial step for a successful protein analysis when only minute amounts are available. While conventional purification methods like dialysis, ultrafiltration or protein precipitation often leads to a marked loss of protein, solid phase extraction (SPE) with small-sized particles is a powerful alternative. The implementation of particles with superparamagnetic cores facilitates the handling of those particles and allows the application of particles in the nm- to low ìm range.
Due to the small diameters, magnetic particles are advantageous for increasing sensitivity when using subsequent MS analysis or gel electrophoresis. In the last years, different types of magnetic particles were developed for specific protein purification purposes followed by analysis or screening procedures using mass spectrometry or SDS gel electrophoresis. In this review, the use of magnetic particles for different applications, like e.g. the extraction and analysis of DNA/RNA, peptides and proteins, is described.
