Download the Free Unbound MEDLINE PubMed App to your smartphone or tablet.
Available for iPhone, iPad, iPod touch, and Android.
Molecular cellular proteomics [journal]
- MS-Viewer: A Web Based Spectral Viewer for Proteomics Results. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Mar 3.
The sharing and viewing of peptide identification results from search engines analyzing mass spectrometry based proteomic data is made difficult by the range of analysis tools employed, in that each produces a different output format. Annotated results associated with a journal article are often required to be made available, but providing these in a format that can be queried by other researchers is often difficult. This is because while standard formats for results have been developed, these are not necessarily easy to produce. In this manuscript we describe the MS-Viewer program, part of the Protein Prospector web package, which uses easy-to-create tabular files as input format for providing highly interactive viewing of search engine results. Due to the simplicity and flexibility of the input format, results from a wide variety of search engines have been successfully viewed through the web interface of this tool.
- Isolation and proteomic characterization of the mouse sperm acrosomal matrix. [Journal Article]
- Mol Cell Proteomics 2014 Mar; 13(3):918.
- The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 27.
The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate (PI3P) to phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). Dysfunctional Vac14 mutants, deficiency of one of the Vac14 complex components or inhibition of PIKfyve enzymatic activity result in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved is only poorly understood. Here we show that ectopic overexpression of wildtype Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal and autophagy-associated proteins. However, only late endosomal marker proteins are bound to the membrane of these enlarged vacuoles. To decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with the multidimensional protein identification technology (MudPIT) was conducted and unraveled novel molecular links. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that regulation of vesicular transport processes as well as phosphatidylinositol modifying enzymes are tightly interconnected.
- SORD overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: A quantitative proteomics study. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 24.
Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated by OFFGEL electrophoresis, and analyzed by LC-coupled tandem MS. Non-redundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples, and cancer cell lines from HCEC cells. Two hundred twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change vs. normal mucosa +/-1.4), which correlated (r=0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (~25%) proteins displayed directionally similar expression changes in colorectal cancer cells (vs. HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein upregulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase (SORD), a key enzyme in the polyol pathway. Validation studies revealed dramatically increased SORD concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism may represent a novel facet of the metabolome remodeling associated with tumorigenesis.
- Phenol Soluble Modulin Variants of Community-Associated Methicillin-Resistant Staphylococcus aureus Captured Using Mass Spectrometry-Based Molecular Networking. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 24.
Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA) possesses more than 2,000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows MRSA with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated MRSA strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin (PSM) family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP 1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other PSM truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single PSM parent peptide can functionally spawn differential bioactivities in response to various external stimuli.
- Quantitative, Time-Resolved Proteomic Analysis by Combining Bioorthogonal Noncanonical Amino Acid Tagging and Pulsed Stable Isotope Labeling by Amino Acids in Cell Culture. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 21.
An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30-min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.
- Improved Normalization of Systematic Biases Affecting Ion Current Measurements in Label-free Proteomics Data. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 21.
Normalization is an important step in the analysis of quantitative proteomics data. If this step is ignored, systematic biases can lead to incorrect assumptions about regulation. The statistical procedures for normalizing proteomics data have been largely borrowed from genomics where their development has focused on the removal of so-called batch effects. In general, a typical normalization step in proteomics works under the assumption that most peptides/proteins do not change; scaling is then used to give a median log-ratio of 0. The focus of this work was to identify other factors, derived from knowledge of the variables in proteomics, which might be used to improve normalization. In this work, we have examined the multi-laboratory datasets from Phase I of the NCI CPTAC program. Surprisingly, the largest sources of bias affecting peptide intensities within labs were retention time and charge state. These effects were exaggerated in samples of unequal concentrations or spike-in levels, presumably because the average precursor charge for peptides with higher charge state potentials is lower at higher relative sample concentrations. These effects are consistent with reduced protonation during electrospray and demonstrate that the physical properties of the peptides themselves can serve as good reporters of systematic biases. Between labs, retention time, precursor m/z, and peptide length were most commonly the top-ranked bias variables, over the standardly used average intensity (A). A larger set of variables was then used to develop a stepwise normalization procedure. This statistical model was found to perform as well or better on the CPTAC mock biomarker data than other commonly used methods. Furthermore, the described method does not require a priori knowledge of the systematic biases in a given dataset. Key improvements here can be attributed to the inclusion of variables other than relative intensity during normalization.
- Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 21.
The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression level is not altered in expression, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase.
- EPASIS: Elution profile analysis of SDS-induced sub-complexes by quantitative mass spectrometry. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 21.
Analyzing the molecular architecture of native multi-protein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five sub-modules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the sub-structure of large multi-protein complexes in a highly time- as well as cost-effective manner.
- An Unbiased Proteomic Screen Reveals Caspase Cleavage is Positively and Negatively Regulated by Substrate Phosphorylation. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Feb 20.
Post-translational modifications of proteins regulate diverse cellular functions with mounting evidence suggesting that hierarchical crosstalk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death by cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins where caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins, finding that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, while cleavage of MST3 is promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7 and -8 recognition motifs, and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage, and that N-terminomic strategies can be tailored to study crosstalk between phosphorylation and proteolysis.