SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*
| Autor: | Yan, Tianyang, Desai, Heta S, Boatner, Lisa M, Yen, Stephanie L, Cao, Jian, Palafox, Maria F, Jami-Alahmadi, Yasaman, Backus, Keriann M |
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| Rok vydání: | 2021 |
| Předmět: |
Proteomics
solid-phase-enhanced sample-preparation high-field asymmetric waveform ion mobility spectrometry Cycloaddition Reaction Human Genome Organic Chemistry single-pot Biotin MS chemoproteomics LC-MS Iodoacetamide Medicinal and Biomolecular Chemistry HEK293 Cells Ion Mobility Spectrometry Genetics Humans Cysteine Biochemistry and Cell Biology LC-MS/MS Peptides cysteine Solid-Phase Synthesis Techniques |
| Zdroj: | Chembiochem : a European journal of chemical biology, vol 22, iss 10 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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