Dataset Information

Proteome-wide detection of cysteine nitrosylation targets and motifs using bio-orthogonal cleavable linker based enrichment and switch technique (Cys-BOOST)

ABSTRACT: Cysteine (Cys) reversible post-translational modifications (PTMs) are emerging as important players in cellular signaling and redox homeostasis. Here we present Cys-BOOST a novel strategy for LC-MS/MS based quantitative analysis of reversibly modified Cys using switch technique, enrichment via bio-orthogonal cleavable linker and quantification using tandem mas tag (TMT) reagents. We performed direct comparison of Cys-BOOST (n=3) with iodo-TMT (n=3) by analyzing the total CysCys from HeLa cell extracts. As a result higher sensitivity (25,019 vs 9,966 Cys peptides), specificity (98 vs 74 %) and technical reproducibility were obtained by Cys-BOOST. In addition, the application of Cys-BOOST for the analysis of Cys nitrosylation (SNO) in S-nitrosoglutathione (GSNO) treated and non-treated HeLa cell extracts lead to the identification of unprecedented number of SNO proteins (3,537), SNO peptides (9,314) and unique SNO sites (8,304). Based on the quantitative data we describe SNO consensus motifs for endogenous SNO and SNO sites with differential reactivity to GSNO. Collectively, our findings suggest Cys-BOOST as a concurrent method of choice for Cys PTM analysis.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ruzanna Mnatsakanyan

LAB HEAD: René P. Zahedi

PROVIDER: PXD011131 | Pride | 2019-05-21

REPOSITORIES: Pride

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Publications

Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique.

Mnatsakanyan Ruzanna R Markoutsa Stavroula S Walbrunn Kim K Roos Andreas A Verhelst Steven H L SHL Zahedi René P RP

Nature communications 20190516 1

Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treate ...[more]

PMID: 31097712

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Project description:Objectives and goals: The causes and molecular pathology of ovarian cancer are essentially unknown. However, it is generally understood that serous ovarian borderline tumors (SBOT) and well differentiated (WD) serous ovarian carcinomas (SC) have a similar tumorigenetic pathway, distinct from moderately (MD) and poorly differentiated (PD) SC. The aim of this study was to identify mRNAs differentially expressed between MD/PD SC, SBOT and superficial scrapings from normal ovaries (SNO),and to correlate these mRNAs with clinical parameters. Results: From the global gene expression analyses, thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected and validated by RT-qPCR, verifying 21 mRNAs to be significantly differentially expressed (p<0.01). Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. VEGFA was significantly upregulated (FC=6.1, p=6.0x10-6), and correlated with progression-free survival (p=0.037). ZNF385B was significantly downregulated (FC=-130.5, p=1.2x10-7), and correlated with overall survival (p=0.03). An increased TPX2 (p=0.03) and FOXM1 (p=0.044) expression correlated with optimal normalization of serum CA125 after treatment. Furthermore, we present a common molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and interacting with TP53. Conclusions: We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including VEGFA and ZNF385B, correlating with survival, and FOXM1 and TPX2, correlating with normalization of serum CA125. By the use of oligonucleotid microarrays (Affymetrix U133 Plus 2.0 Arrays) differences in mRNA expression between eleven MD/PD SC, eight SBOT and four SNO samples were analyzed. Differentially expressed mRNAs were selected for validation by RT-qPCR in additional samples totaling 21 MD/PD SC, 13 SBOT and seven samples of SNO. Each mRNA was run in quadruple. The mRNAs differentially expressed in MD/PD SC compared with SNO were correlated with clinical parameters.

Project description:Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate most physio-pathological states. Here, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by enrichment with titanium dioxide (TiO2) and mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to Hela cells lysate, achieving high specificity and enrichment efficiency. In particular,From 500µg of Hela cell lysate, the method led to the identification of 9229 unique Cys peptides starting from 500µg of Hela cell lysate . the CysPAT labelled Cys peptides showed increased hydrophobicity. Finally, the method was further developed to simultaneously enrich Cys peptides and phosphorylated peptidesThe method was further developed to simultaneously enrich Cys peptides and phosphorylated peptides from SILAC labelled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, of 22972 unique reversibly modified Cys peptides (3886 proteins) and 18549 unique phosphopeptides (2257 proteins) were simultaneously identified by Orbitrap Fusion. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins interacted with EGF receptor (EGFR)-binding proteins and proteins involved in EGFR signaling pathway. EGF stimulation can activate the phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), meanwhile, it also leads to the activation of multiple protein tyrosine phosphatases (PTPs) by reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif, Llater the activated PTPs would dephosphorylate and inactivate the molecules activated by EGF stimulation. Overall, the CysPAT strategy is a promising tool for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced cysteine modifications.

Dataset Information

Proteome-wide detection of cysteine nitrosylation targets and motifs using bio-orthogonal cleavable linker based enrichment and switch technique (Cys-BOOST)

Publications

Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique.

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