Chemoproteomic Cartography of Cysteine Modifications

Although cysteine residues are rare in proteins, their reactive thiol group makes them biologically crucial. Cysteines act as redox sensors, catalytic residues, and sites of post-translational modification (PTMs). Dysregulation of cysteine-dependent processes is linked to cancer, neurodegeneration, and inflammatory disorders, making cysteines attractive therapeutic targets. This thesis explores cysteine modifications from biological and chemoproteomic perspectives. Chapter 1 introduces bottom-up mass spectrometry (MS)-based proteomics approaches for cysteine modification analysis and reviews major cysteine PTMs, their biological roles, and chemoproteomic strategies to probe cysteine reactivity. Chapters 2-4 focus on the reversible lipid modification long-chain S-acylation, which regulates protein trafficking, stability, and signaling. Although an estimated 15% of the proteome undergoes this modification and it is implicated in diseases, long-chain S-acylation remains underexplored in drug discovery. A key challenge is limited site-specific knowledge of which cysteines are modified under various biological conditions. Chapter 2 reviews mass spectrometry-based methods to study long-chain S-acylation, including acyl-biotin exchange (ABE), metabolic labeling, zDHHC substrate profiling, and protein-centric approaches. Chapter 3 investigates long-chain S-acylation in resting and pro-inflammatory THP-1 macrophages using quantitative, site-specific ABE. Results reveal S-acylation is dynamic and responsive to inflammatory activation, affecting regulators and newly identified inflammation-associated sites on immune signaling proteins. Proteome-wide evidence for coexisting, site-selectively modified S-acyl-peptidoforms suggests distinct cysteine occupancy patterns may serve as regulatory mechanisms during macrophage polarization. Functional perturbation experiments show that altering the balance between S-acylation and deacylation modulates inflammatory output, establishing long-chain S-acylation as a regulator of innate immunity and a therapeutic target in macrophage-driven inflammation. Chapter 4 examines long-chain S-acylation during aging in C. elegans, generating a site-specific atlas of this modification across the aging proteome. Data show remodeling of S-acylation during adulthood, particularly on proteins involved in proteostasis and lipid metabolism. Analysis of long- and short-lived mutant strains reveals that altered longevity correlates with distinct S-acylation signatures. These findings position long-chain S-acylation as a possible regulatory layer that changes with age and may contribute to aging phenotypes. Chapters 5-6 shift toward cysteine-focused drug discovery. Site-specific small-molecule engagement can determine therapeutic efficacy and safety, making accurate identification of targets and off-targets essential. Competitive chemoproteomic strategies such as activity-based protein profiling (ABPP) enable quantitative, site-resolved analysis of covalent drug-protein interactions but require tailored approaches for certain cysteine subclasses. Chapter 5 introduces thio-BE, a protein-level ABE-based workflow that enables measurement of thioester-linked catalytic intermediates within the ubiquitin conjugation machinery. Applied in HEK293-T cells, thio-BE confirms UBA3, UBE2M, and UBE2F as targets of the UBA3 inhibitor Pevonedistat, demonstrating utility for monitoring small-molecule effects in the thioesterome. Chapter 6 presents isoPhosID, an advanced PhosID-ABPP workflow incorporating isotopically labeled phosphonate handles for automated enrichment and site-specific quantification. Applied in THP-1 monocytes, isoPhosID expands cysteine coverage, enables isoform-specific resolution, and enhances analytical depth through optimized chemistry and protease selection. Overall, this thesis advances methodology and biological insight by enabling site-specific analysis of cysteine modifications and drug-protein interactions. It reveals functional roles for cysteine PTMs in inflammation and aging while expanding the chemoproteomic toolkit for cysteine-targeted therapeutic development.