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

ABSTRACT: Current methods 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 in 18 min per compound. We applied it to identifying the proteome-wide targets of a covalent inhibitor of mutant KRASG12C and of ibrutinib, a covalent inhibitor of BTK. In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines , which includes >20,000 cysteines in >6,000 proteins per cell line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spleen, Epithelial Cell

SUBMITTER: Miljan Kuljanin  

LAB HEAD: Steven P. Gygi

PROVIDER: PXD022511 | Pride | 2021-01-26

REPOSITORIES: Pride