Dataset Information

Rapid and high coverage profile of human cysteineome

ABSTRACT: Cysteines play important roles in many biological activities, including protein folding, aging, signaling and etc. To identify and quantitatively profile the oxidation and reactivity of cysteines, cysteine-reactive compounds have been applied to covalently label and enrich the cysteines. And with mass spectrometry (MS), thousands of cysteines can be profiled at the same time with decoration of functional groups. However, with all the technology and methods developed to characterize cysteines, current datasets have only uncovered about 5% of the total cysteinome. Here we show a comprehensive and high coverage profile of more than 30,000 cysteines. With incorporation of new techniques - single-pot, solid phase-enhanced sample-preparation (SP3) and High-field asymmetric waveform ion mobility spectrometry (FAIMS), we have successfully identified as many as 14,306 cystienes in a single-shot experiment. With cysteine profiles in different cell lines, proteases and fractionations, our results have successfully expanded the cysteine library to a great extent.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tianyang Yan

LAB HEAD: Keriann M. Backus

PROVIDER: PXD023060 | Pride | 2021-02-03

REPOSITORIES: Pride

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Publications

SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*.

Yan Tianyang T Desai Heta S HS Boatner Lisa M LM Yen Stephanie L SL Cao Jian J Palafox Maria F MF Jami-Alahmadi Yasaman Y Backus Keriann M KM

Chembiochem : a European journal of chemical biology 20210218 10

Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhan ...[more]

PMID: 33442901

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Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

Dataset Information

Rapid and high coverage profile of human cysteineome

Publications

SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*.

Similar Datasets

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