Graphical AbstractHighlights d Mutation-phosphorylation correlation suggests possible signaling interplays in EOGCs d mRNA-protein correlation suggests genes with high association with patient survival d Integrated analysis of mRNA and protein data identified four subtypes d Phosphorylation data provide cellular signaling pathways underlying the subtypes SUMMARYWe report proteogenomic analysis of diffuse gastric cancers (GCs) in young populations. Phosphoproteome data elucidated signaling pathways associated with somatic mutations based on mutation-phosphorylation correlations. Moreover, correlations between mRNA and protein abundances provided potential oncogenes and tumor suppressors associated with patient survival. Furthermore, integrated clustering of mRNA, protein, phosphorylation, and N-glycosylation data identified four subtypes of diffuse GCs. Distinguishing these subtypes was possible by proteomic data. Four subtypes were associated with proliferation, immune response, metabolism, and invasion, respectively; and associations of the subtypes with immune-and invasion-related pathways were identified mainly by phosphorylation and N-glycosylation data. Therefore, our proteogenomic analysis provides additional information beyond genomic analyses, which can improve understanding of cancer biology and patient stratification in diffuse GCs.

With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel "multi-blind" spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010199, 1-13, 2012. Post-translational modifications (PTMs)1 regulate protein function, localization, and interactions inside a cell (1). Hundreds of PTM types are known so far, and yet a lot more may remain to be discovered (2, 3). The identification of PTMs is critical to gaining insight into biological functions but remains a formidable challenge. Tandem mass spectrometry (MS/MS) has emerged as a powerful tool for rapid identification of PTMs (4, 5), which can be detected by PTM-related diagnostic mass shifts of fragment ions in MS/MS spectra. However accurate computational identification of modified peptides remains a difficult problem often addressed with restrictive approaches that require "guessed" lists of possible PTMs to be provided in advance (6 -8). Such an approach may overlook potentially important PTMs if they are not guessed in advance. In recent PTM identification algorithms, peptide sequence tag approaches have been proposed to search for more types of PTMs and to speed up the search (9 -12). A small set of short sequence tags (2-4 amino acids long) are derived from an MS/MS spectrum and used to screen for matching peptides in a protein database; possible modifications are then inferred from the difference between the precursor ion mass of the experimental spectrum and the theoretically calculated mass of the matched peptide.In contrast with restrictive approaches, unrestrictive or blind approaches search MS/MS spectra for all known and even possibly unknown types of PTMs at once and derive the list of modifications directly from MS/MS data (13-22). OpenSea (13) and SPIDER (14) compared de novo sequencing results with peptide sequences from a protei...

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

Isoform-specific signaling of Akt, a major signaling hub and a prominent therapeutic target, remained poorly defined until recently. Subcellular distribution, tissue-specific expression, substrate specificity, and posttranslational modifications are believed to underlie isoform-specific signaling of Akt. The studies reported here show inhibition of Akt2 activity under physiologically relevant conditions of oxidation created by PDGF-induced reactive oxygen species. Combined MS and functional assays identified Cys124 located in the linker region between the N-terminal pleckstrin homology domain and the catalytic kinase domain as one of the unique regulatory redox sites in Akt2 with functional consequence on PDGF-stimulated glucose uptake. A model is proposed describing the consequence of increased endogenous oxidation induced by extracellular cues such as PDGF on Akt2 activity.disulfide | receptor tyrosine kinase | DCF | DCP-Bio1 P KB/Akt is a major signaling hub between cytokine, growth factor, and integrin signaling pathways of consequence to many biological processes. Energy storage, protein synthesis, cell survival and growth, cell cycle progression, and cell death are differentially regulated by the three known isoforms of Akt kinase: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ (1). Although the molecular features underlying isoform-specific functional predominance are largely unknown, hypotheses to explain Akt isoform specificity include selective interactions with substrates and/ or binding partners, tissue specificity, subcellular location, and temporal changes in activation profiles of Akt isoforms (2). Posttranslational modifications may contribute to this isoform-specific signaling, but reports of modifications are lacking.Reactive oxygen species (ROS) are integral to cytokine and growth factor signaling; ROS generation in response to these extracellular cues is well documented (3). Earlier reports, in particular from Sundaresan et al. (4), showed ROS generation in response to PDGF stimulation of vascular smooth muscle cells and suggested that H 2 O 2 can relay redox signals to regulate physiological signaling in response to growth factors. More recently, it has been shown that PrxI phosphorylation at Y194 by Src family tyrosine kinases inhibited PrxI and resulted in H 2 O 2 accumulation at the cellular membrane where receptor tyrosine kinase activation occurs (5). Unanswered questions include which proteins are oxidized by receptor tyrosine kinase-induced ROS, which specific cysteine site(s) undergo oxidation, and the consequence of oxidation on activity of these proteins and propagation of receptor tyrosine kinase signaling. Recently, the synthesis of several dimedone-based chemoselective reagents capable of specific labeling of sulfenic acid oxidized proteins was reported; these reagents allow for specific enrichment and identification of oxidized proteins (6-10). In this study, we used a biotin-tagged 1,3-cyclohexadione derivative, DCP-Bio1 (Fig. 1A), to investigate isoform-specific effects of PDGF-induced ...

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel lowabundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MOD i and MODmap algorithm. We observed unexpected mass shifts (⌬m ‫؍‬ ؊16, ؊34, ؉64, ؉87, and ؉103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of ؊16, ؊34, and ؉64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO 2 -SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO 2 -Cys) either to DHA (⌬m ‫؍‬ ؊34 Da) or serine along with Cys-SO 2 -SH (⌬m ‫؍‬ ؉64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (؉87 and ؉103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO 2 -SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.