Accelerating multiplexed profiling of protein-ligand interactions: high-throughput plate-based reactive cysteine profiling with minimal input

ABSTRACT: Chemoproteomics investigates small molecule-protein interactions and has made significant progress in recent years. Despite its vast potential, the proteome-wide profiling of reactive cysteine ligandability remains a formidable task to adapt for high throughput applications. This is primarily due to a lack of platforms capable of achieving the desired depth using low sample input in 96- or 384-well plates. Here we have revamped the cysteine profiling platform to address the challenge with an eye toward performing high-throughput library screening in plates. By incorporating several changes including i) an 18-plex TMT sample multiplexing strategy, ii) a magnetic beads-based one-pot workflow, iii) a 10X higher capacity streptavidin resin, and iv) optimized mass spectrometry analyses, a plate-based platform was developed that enables routine interrogation of either ~18,000 or ~24,000 reactive cysteines based on starting amounts of 10 or 20 µg, respectively. We applied the platform to screen a library of 192 electrophiles in the native HEK293T proteome, mapping the ligandablity of 38,450 reactive cysteines from 8,274 human proteins. The significantly improved depth revealed many previously unknown reactive cysteines and cysteine-ligand interactions and led to the identification of an azepane-containing acrylamide which has preferential binding to cysteines in EGF-like domains. We further applied the platform to characterize new cellular targets of well-studied compounds and covalent drugs in three different human cell lines. We found that ARS-1620, a KRASG12C inhibitor, also binds to cysteine 140 of an off-target adenosine kinase ADK, inhibiting its kinase activity. The platform represents a major step forward to high throughput evaluation of reactive cysteines on a proteome-wide scale.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Qing Yu  

LAB HEAD: Steven Gygi

PROVIDER: PXD044402 | Pride | 2023-12-24

REPOSITORIES: Pride