Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with singlepot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line highfield asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of > 14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only~28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.
Mass spectrometry-based chemoproteomics
has enabled functional
analysis and small molecule screening at thousands of cysteine residues
in parallel. 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) or “click chemistry” for cysteine
capture. Despite considerable advances in both sample preparation
and analytical platforms, current techniques only sample a small fraction
of all cysteines encoded in the human proteome. Extending the recently
introduced labile mode of the MSFragger search engine, here we report
an in-depth analysis of cysteine biotinylation via click chemistry
(CBCC) reagent gas-phase fragmentation during MS/MS analysis. We find
that CBCC conjugates produce both known and novel diagnostic fragments
and peptide remainder ions. Among these species, we identified a candidate
signature ion for CBCC peptides, the cyclic oxonium-biotin fragment
ion that is generated upon fragmentation of the N(triazole)–C(alkyl)
bond. Guided by our empirical comparison of fragmentation patterns
of six CBCC reagent combinations, we achieved enhanced coverage of
cysteine-labeled peptides. Implementation of labile searches afforded
unique PSMs and provides a roadmap for the utility of such searches
in enhancing chemoproteomic peptide coverage.
Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis, together with unexpected non-organelle specific TurboID-catalyzed proximity labeling. This mislabeling was minimized through simultaneous depletion of both endogenous biotin and newly translated TurboID fusion protein. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.
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