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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

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.

  • 4 supplemental files
Cover page: Novel Approaches for Enhanced Chemoproteomic Profiling of the Human Cysteinome
  • Article
  • Peer Reviewed
UCLA Previously Published Works (2024)
Pinpointing functional, structural, and redox-sensitive cysteines is a central challenge of chemoproteomics. Here, we present a protocol comprising two dual-enrichment cysteine chemoproteomic techniques that enable capture of cysteines (Cys-LoC) and quantification of cysteine oxidation state (Cys-LOx) in a localization-specific manner. We describe steps for utilizing TurboID-mediated protein biotinylation for enrichment of compartment-specific proteins, followed by click-mediated biotinylation and enrichment of cysteine-containing peptides. Thus, changes to compartment-specific cysteine identification and redox state can be assessed in a variety of contexts. For complete details on the use and execution of this protocol, please refer to Yan et al. (2023).1.
    Cover page: Protocol for organelle-specific cysteine capture and quantification of cysteine oxidation state.
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2023)
    The plasma membrane proteome is a rich resource of functionally important and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here, we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of low-density lipoprotein (LDL) particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.
      Cover page: Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2022)
      Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. 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.
        Cover page: SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2022)
        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.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2021)
          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 single-pot, 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 high-field 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.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2023)
            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, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we 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. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.
              Cover page: Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
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