Tandem mass spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared among multiple proteins. IDPicker is an open-source protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified False Discovery Rate. Here, we update IDPicker to increase confident peptide identifications by combining multiple scores produced by database search tools. By segregating peptide identifications for thresholding using both the precursor charge state and the number of tryptic termini, IDPicker retrieves more peptides for protein assembly. The new version is more robust against false positive proteins, especially in searches using multispecies databases, by requiring additional novel peptides in the parsimony process. IDPicker has been designed for incorporation in many identification workflows by the addition of a graphical user interface and the ability to read identifications from the pepXML format. These advances position IDPicker for high peptide discrimination and reliable protein assembly in large-scale proteomics studies. The source code and binaries for the latest version of IDPicker are available from

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the inva-

The standard shotgun proteomics data analysis strategy relies on searching MS/MS spectra against a context-independent protein sequence database derived from the complete genome sequence of an organism. Because transcriptome sequence analysis (RNA-Seq) promises an unbiased and comprehensive picture of the transcriptome, we reason that a sample-specific protein database derived from RNA-Seq data can better approximate the real protein pool in the sample and thus improve protein identification. In this study, we have developed a two-step strategy for building sample-specific protein databases from RNA-Seq data. First, the database size is reduced by eliminating unexpressed or lowly expressed genes according to transcript quantification. Secondly, high-quality nonsynonymous coding single nucleotide variations (SNVs) are identified based on RNA-Seq data, and corresponding protein variants are added to the database. Using RNA-Seq and shotgun proteomics data from two colorectal cancer cell lines SW480 and RKO, we demonstrated that customized protein sequence databases could significantly increase the sensitivity of peptide identification, reduce ambiguity in protein assembly, and enable the detection of known and novel peptide variants. Thus, sample-specific databases from RNA-Seq data can enable more sensitive and comprehensive protein discovery in shotgun proteomics studies.

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics. DNA sequence variation is associated with diseases and differential drug response. As a paradigmatic example, cancers are diseases of clonal proliferations caused by mutations in oncogenes and tumor suppressor genes (1). After several decades of searching through traditional biology approaches, many mutant genes have been causally implicated in oncogenesis (2). Facilitated by the new genomic techniques such as SNP (single nucleotide polymorphism) arrays and deepsequencing, the identification of cancer genes has made enormous progress over the past several years (3-7). The genomic abnormalities of cancer are expressed through aberrant proteins and proteomes and their altered functions. Although proteins reflecting the genomic changes in cancer have the potential to become clinically meaningful biomarkers, their discovery and validation has proven to be challenging. As a result, few biomarker candidates have translated into clinical use.Over the past decade, mass spectrometry (MS)-based shotgun proteomics has emerged as a high-throughput, unbiased method for the identification of proteins in complex samples (8,9). Its application to tumor specimens holds great potential in identifying mutant proteins in human cancers. However, because sh...

Atherosclerosis is a complex process including proinflammatory and pro-oxidative events, which recruit monocytes and lymphocytes to adhere to the surface of the injured endothelium as the first observable event. Epidemiological studies have revealed numerous risk factors for atherosclerosis including genetic and environmental risk factors, such as smoking, diet, infection, and air pollution, which interestingly have also been known to induce oxidative stress.The thioredoxin-1 (Trx1) system plays a key role in modulating redox signaling pathways regulating physiological as well as pathophysiological processes such as atherosclerosis development.1,2 Trx1 is a major substrate for thioredoxin reductase-1 (TrxR1), serving as an electron carrier to reduce peroxiredoxins, redox factor-1 (Ref1), and other proteins. A dithiol (Cys-32, Cys-35) in the active site of Trx1 undergoes reversible oxidation to the disulfide during the transfer of reducing equivalents. In addition to the redox regulatory function of the active-site cysteines (Cys-32, Cys-35), post-translational modifications of the other cysteines by oxidation (Cys-62, Cys-69), S-nitrosylation (Cys-69), and glutathionylation (Cys-73) have a significant effect on Trx1 function.3-5 Modification of thiols in Trx1 interrupts signaling mechanisms involved in cell growth, proliferation, and apoptosis. 3,4 Previous studies show that common products of lipid peroxidation, including acrolein and 4-hydroxy-2-nonenal (HNE) react with and modify functions of various cellular proteins. Acrolein and HNE are electrophilic lipids that target a number of redox-sensitive proteins, inducing multiple cellular responses through several mechanisms. 6 The cytoprotective role of acrolein and HNE in cellular mechanisms has been demonstrated at low concentrations. For example, HNE induces cytoprotective antioxidants, HO-1 and glutathione (GSH), in bovine aortic endothelial cells (BAECs) 7 and activates thioredoxin

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Summary A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We employed standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data. Biological replicate cultures yielded highly consistent proteomic inventories with a cumulative total of 6,513 protein groups with a protein FDR of 3.17% across all cell lines. Networks of co-expressed proteins with differential expression based on MMR status revealed impact on protein folding, turnover and transport, on cellular metabolism and on DNA and RNA synthesis and repair. Analysis of variant amino acid sequences suggested higher stability of proteins affected by naturally occurring germline polymorphisms than of proteins affected by somatic protein sequence changes. The data provide evidence for multi-system adaptation to MMR deficiency with a stress response that targets misfolded proteins for degradation through the ubiquitin-dependent proteasome pathway. Enrichment analysis suggested epithelial-to-mesenchymal transition (EMT) in RKO cells, as evidenced by increased mobility and invasion properties compared to SW480. The observed proteomic profiles demonstrate previously unknown consequences of altered DNA repair and provide an expanded basis for mechanistic interpretation of MMR phenotypes.