- A strong neutrophil elastase proteolytic fingerprint marks the carcinoma tumor proteome. [Journal Article]
- MCMol Cell Proteomics 2016 Dec 07
- Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic datasets representative of colon adenocarcinoma (AC)...
Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic datasets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint composed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly overrepresented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent cancer-specific proteolytic fingerprint detected. This sequence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large fraction of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific peptides that are possible markers of tumor-infiltrating neutrophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Dataset 1) and PXD004249 (Dataset 2). Our results indicate the value of peptide-wise analysis of large global proteomic analysis datasets as opposed to protein-wise analysis, in which outlier differential peptides are usually neglected.
- Accounting for protein subcellular localization: A compartmental map of the rat liver proteome. [Journal Article]
- MCMol Cell Proteomics 2016 Dec 06
- Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cel...
Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle while a significant fraction may reside elsewhere. As a step towards a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this dataset, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at six months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene.
- A molecular basis for the presentation of phosphorylated peptides by HLA-B antigens. [Journal Article]
- MCMol Cell Proteomics 2016 Dec 05
- As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of...
As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39 or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its non-phosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.
- A proteomic approach to analyse the aspirin-mediated lysine acetylome. [Journal Article]
- MCMol Cell Proteomics 2016 Dec 02
- Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. As...
Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino-acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences non-specific chemical acetylations.
- Human spermatozoa quantitative proteomic signature classifies normo- and asthenozoospermia. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 28
- Scarcely understood defects lead to asthenozoospermia which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interven...
Scarcely understood defects lead to asthenozoospermia which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma dataset which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the dataset. It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples. This is despite the results that some of the seminal plasma proteins have big fold changes among classes but they fall short of statistical significance. S-Plot of the sperm proteomic dataset generated some high confidence targets which might be implicated in sperm motility pathways. These proteins also had the area under the curve value of 0.9 or 1 in ROC curve analysis. Various pathways were either enriched in these proteomic datasets by pathway analysis or they were searched by their constituent proteins. Some of these pathways were axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly among others. The mass spectrometric data is available via ProteomeXchange with identifier PXD004098.
- CSF-PR 2.0: an interactive literature guide to quantitative cerebrospinal fluid mass spectrometry data from neurodegenerative disorders. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 27
- The rapidly growing number of biomedical studies supported by mass spectrometry based quantitative proteomics data has made it increasingly difficult to obtain an overview of the current status of th...
The rapidly growing number of biomedical studies supported by mass spectrometry based quantitative proteomics data has made it increasingly difficult to obtain an overview of the current status of the research field. A better way of organizing the biomedical proteomics information from these studies and making it available to the research community is therefore called for. In the presented work, we have investigated scientific publications describing the analysis of the cerebrospinal fluid proteome in relation to multiple sclerosis, Parkinsons disease and Alzheimers disease. Based on a detailed set of filtering criteria we extracted 85 datasets containing quantitative information for close to 2000 proteins. This information was made available in CSF-PR 2.0 (http://probe.uib.no/csf-pr-2.0), which includes novel approaches for filtering, visualizing and comparing quantitative proteomics information in an interactive and user-friendly environment. CSF-PR 2.0 will be an invaluable resource for anyone interested in quantitative proteomics on cerebrospinal fluid.
- Multi-omics analysis of serum samples demonstrates reprogramming of organ functions via systemic calcium mobilization and platelet activation in metastatic melanoma. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 22
- Pathophysiologies of cancer-associated syndroms such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including...
Pathophysiologies of cancer-associated syndroms such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44 and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alterate levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.
- E. coli proteome microarrays identified the substrates of ClpYQ protease. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 18
- Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases play a crucial role within this process. In Escherichia coli, ClpYQ is one of t...
Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases play a crucial role within this process. In Escherichia coli, ClpYQ is one of the primary ATP-dependent proteases. In addition to function with removals of abnormal peptides in the cells, ClpYQ degrades regulatory proteins if necessary and thus let cells adjust to various environmental conditions. In E. coli, SulA, RcsA, RpoH and TraJ as well as RNase R, have been identified as natural protein substrates of ClpYQ. ClpYQ contains ClpY and ClpQ. The ATPase ClpY is responsible for protein recognition, unfolding, and translocation into the catalytic core of ClpQ. In this study, we use an indirect identification strategy to screen possible ClpY targets with E. coli K12 proteome chips. The chip assay results showed that YbaB strongly bound to ClpY. We used yeast two-hybrid assay to confirm the interactions between ClpY and YbaB protein and determined the Kd between ClpY and YbaB by quartz crystal microbalance. Furthermore, we validated that YbaB was successfully degraded by ClpYQ protease activity using ClpYQ in vitro and in vivo degradation assay. These findings demonstrated the YbaB is a novel substrate of ClpYQ protease. This work also successfully demonstrated that with the use of recognition element of a protease can successfully screen its substrates by indirect proteome chip screening assay.
- Quantitative proteomic analysis of replicative and non-replicative forms reveals important insights into chromatin biology of Trypanosoma cruzi. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 16
- Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and non-r...
Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and non-replicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and non-replicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and non-chromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in non-replicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the non-replicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in non-replicative forms, but located at non-chromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and non-chromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Since trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types.
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- Quantitative GTPase affinity purification identifies Rho family protein interaction partners. [Journal Article]
- MCMol Cell Proteomics 2016 Nov 16
- Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop qu...
Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, RhoD) depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors and mirrors evolutionary relationships among Rho GTPases.