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SP3‐FAIMS Chemoproteomics for High‐Coverage Profiling of the Human Cysteinome **
Author(s) -
Yan Tianyang,
Desai Heta S.,
Boatner Lisa M.,
Yen Stephanie L.,
Cao Jian,
Palafox Maria F.,
JamiAlahmadi Yasaman,
Backus Keriann M.
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000870
Subject(s) - chemistry , ion mobility spectrometry , proteome , human proteome project , small molecule , biotinylation , computational biology , sample preparation , druggability , chromatography , proteomics , computer science , combinatorial chemistry , mass spectrometry , biochemistry , biology , gene
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‐enhanced sample‐preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on‐line high‐field asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single‐shot 70 min experiment. Showcasing the wide utility of the SP3‐FAIMS chemoproteomic method, we find that it is also compatible with competitive small‐molecule screening by isotopic tandem orthogonal proteolysis–activity‐based protein profiling (isoTOP‐ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3‐FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.