UCLA

Cysteine-directed chemoproteomic profiling methods yield high-throughput inventories of redox-sensitive and ligandable cysteine residues. They are enabling techniques for functional biology. Due to their nucleophilicity and sensitivity to alkylation, cysteines have emerged as attractive sites to target with chemical probes. Cysteine-reactive covalent compounds can access small and poorly defined binding sites and efficiently block high-affinity interactions or compete with high concentrations of endogenous biomolecules. Furthermore, cysteine is the most frequently acquired amino acid due to missense variants in cancer databases. Acquired cysteines are both driver mutations and sites targeted by precision therapies; however, despite their ubiquity, nearly all acquired cysteines remain uncharacterized. Regardless of improvements in sample preparation workflows, cysteine chemoproteomic experiments still only sample a small fraction of the human cysteinome due to biological factors such as protein abundance, restricted protein expression profiles, and technical factors such as unoptimized data analysis workflows not tailored to chemoproteomics, including database searches that do not sample the mutation-induced variant proteome. The cumbersome nature of these sample preparation workflows along with reagent costs hinder most chemoproteomics studies. In this work, we develop two new chemoproteomics platforms to enable high-throughput identification of redox sensitive and ligandable cysteines, including gain-of-cysteines. First, we tailor our single-pot, solid-phase-enhanced sample preparation (SP3) method to specifically probe the redox proteome, which showcases the utility of the SP3 platform in multistep sample-preparation workflows. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation. By implementing a customized workflow in the FragPipe computational pipeline, we achieve accurate MS1-based quantification, including for peptides containing multiple cysteine residues. We also present “chemoproteogenomics”, combining proteogenomics with established chemoproteomics methods to study human missense variation resulting in neo cysteine residues or mutations nearby cysteine residues. For both cancer and healthy genomes, we find that cysteine acquisition is a ubiquitous consequence of genetic variation that is further elevated in the context of decreased DNA repair. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized 2-stage false discovery rate (FDR) error controlled proteomic search enhanced with a user-friendly FragPipe interface to improve coverage of acquired cysteine variants and proximal variants using a panel of 11 cancer cell lines. These two established pipelines allow us to extend activity-based profiling methods, including small molecule screening and redox-profiling, to gain-of-cysteine variants and cysteines proximal to variants. We expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.