UCLA

Mass spectrometry (MS)-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Widely adopted chemoproteomic sample preparation workflows rely on the use of pan cysteine-reactive probes such as iodoacetamide alkyne combined with biotinylation via copper-catalyzed azide-alkyne cycloaddition (CuAAC) for cysteine capture. Despite widespread adoption and considerable advances in both workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. In this work, novel approaches have been developed for enhanced chemoproteomic profiling of the human cysteinome, spanning optimized sample-preparation workflow, in-depth analysis of MS fragmentation products and novel platforms targeting subcellular specific cysteines. First, we developed an optimized sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-preparation (SP3) and on-line high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides. We achieved unprecedented coverage of 34,225 unique cysteines using only ∼28 h of instrument time. Next, we reported an in-depth analysis of cysteine biotinylation via click chemistry (CBCC) reagent gas-phase fragmentation during MS/MS analysis. We found several diagnostic fragments and peptide remainder ions for CBCC peptides. Implementation of labile searches regarding these ions afforded unique peptide spectrum matches (PSMs). Then, we focused on profiling localization-dependent cysteine activities, which bulky proteomic analysis failed to capture. We established the local cysteine capture (Cys-LoC), and local cysteine oxidation (Cys-LOx) methods, which together yielded compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated macrophages revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation. We also developed a novel dual enrichment method to achieve chemoproteomic profiling of cell surface cysteines (Cys-Surf). Our Cys-Surf' platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including residues with oxidation states sensitive to exogenous reductants and changes in cellular activation state. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 529 total ligandable cysteines, including known and novel sites, with an activity-based-protein profiling readout substantiating the ligandability of HLA-A Cys 363. Altogether, this work provided a solid foundation for future studies aiming at deciphering functions and druggability of the human cysteineome.