Dataset Information

PChem_benchmarking_dataset_QTRP_diff_instruments

ABSTRACT: Data sets are used for benchmarking, pChem, a modification-centric, blind-search tool to provide a streamlined pipeline for unbiased assessing of the performance of chemoproteomic probes. The IPM-based QTRP (quantitative thiol reactivity profiling) data sets were recorded on different LC-MS/MS instruments from three vendors. IPM is a clickable thiol reactive reagent with the iodoacetamide warhead.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: jixiang he

LAB HEAD: Jing Yang

PROVIDER: PXD027758 | Pride | 2022-12-16

REPOSITORIES: Pride

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			Action	DRS
	5600_Yangjing_hejixiang_IPM_1ug_75min_20210324-2000ng.xlsx	Xlsx
	5600_Yangjing_hejixiang_IPM_1ug_75min_20210324.wiff	Wiff
	5600_Yangjing_hejixiang_IPM_1ug_75min_20210324.wiff.scan	Wiff
	6600_YangJ_60min_IPM_1_10ulmin_top40_3ug_20210707.wiff	Wiff
	6600_YangJ_60min_IPM_1_10ulmin_top40_3ug_20210707.wiff.scan	Wiff

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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

PChem_benchmarking_dataset_QTRP_diff_instruments

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