Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.

ABSTRACT: Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)

SUBMITTER: Kuljanin M 

PROVIDER: S-EPMC8316984 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature