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Enriching cysteine-containing peptides using a sulfhydryl-reactive alkylating reagent with a phosphonic acid group, and immobilized metal affinity chromatography

ABSTRACT: The reduction of disulfide bonds and their subsequent alkylation is commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of SH-SY5Y cells following 24 hr treatments with two proteasome inhibitors (bortezomib and MG-132) in a TMTpro9-plex experiment. We acquired three datasets - 1) Cys-enriched, 2) Cys-depleted, and 3) non-depleted - and compared the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-CysPAT quantified over 38,000 Cys-containing peptides in 5 hr with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9,900 protein abundance profiles that exhibit the effects of two different proteosome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this “Mini-Cys” dataset can be used to preview and assess the quality of a deep, fractionated dataset.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD038382 | Pride | 2023-12-28

REPOSITORIES: Pride

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Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent with a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography.

Liu Xinyue X Rossio Valentina V Gygi Steven P SP Paulo Joao A JA

Journal of proteome research 20230327 4

The reduction of disulfide bonds and their subsequent alkylation are commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of the SH-SY5Y human cell line following 24 h treatments with two proteasome inhibitors (bortezo ...[more]

PMID: 36971515

Similar Datasets

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.

Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

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