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

Autor: Yan T; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.; Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA., Desai HS; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.; Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA., Boatner LM; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.; Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA., Yen SL; Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA., Cao J; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA., Palafox MF; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.; Department of Human Genetics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA., Jami-Alahmadi Y; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA., Backus KM; Department of Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.; Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA.; Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA.; DOE Institute for Genomics and Proteomics, UCLA, Los Angeles, CA 90095, USA.; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA.; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2021 May 14; Vol. 22 (10), pp. 1841-1851. Date of Electronic Publication: 2021 Feb 18.
DOI: 10.1002/cbic.202000870
Abstrakt: 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.
(© 2021 Wiley-VCH GmbH.)
Databáze: MEDLINE
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