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Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries
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Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries
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Journal Article
Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries
2021
Overview
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)
G12C
and Bruton’s tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
An improved workflow enables a 42-fold higher throughput of activity-based protein profiling.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ Agammaglobulinaemia Tyrosine Kinase - genetics
/ Antioxidant Response Elements - genetics
/ Biomedical and Life Sciences
/ Biomedical Engineering/Biotechnology
/ Cysteine
/ High-throughput screening (Biochemical assaying)
/ Humans
/ Kinases
/ Methods
/ Proteins
/ Proto-Oncogene Proteins p21(ras) - genetics
/ Resource
/ Sarcoma
/ Tyrosine
/ Workflow
