PXD016048

PXD016048 is an original dataset announced via ProteomeXchange.

Title	Quantitative thiol reactivity profiling
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.
HostingRepository	PRIDE
AnnounceDate	2020-07-24
AnnouncementXML	Submission_2020-07-23_22:59:29.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ke Liu
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-10-28 03:59:45	ID requested
⏵ 1	2020-07-23 22:59:30	announced

Fu L, Li Z, Liu K, Tian C, He J, He J, He F, Xu P, Yang J, A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes. Nat Protoc, 15(9):2891-2919(2020) [pubmed]

submitter keyword: Thiol, Redox, Electrophile, Chemical proteomics

Jing Yang
contact affiliation	State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Lifeomics, 102206 Beijing, China
contact email	yangjing54@hotmail.com
lab head
Ke Liu
contact affiliation	BPRC
contact email	liukekeno1@126.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD016048
     1. Label: PRIDE project
     2. Name: Quantitative thiol reactivity profiling