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An electrophilic arginine mimetic, 2-chloroacetamidine (CAM), was deployed to enable trypsin-mediated proteolysis at cysteine residues and to enhance mass spectrometry-based proteomic detection of cysteine-containing peptides. Illustrating the value of the CAM-capping strategy, proteogenomic analysis using a two-stage false discovery rate (FDR) search revealed >50% enhanced coverage of missense variants, when compared to established workflows.
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Cisteína , Tripsina , Cisteína/química , Tripsina/metabolismo , Tripsina/química , Acetamidas/química , Proteolisis , Proteómica , Péptidos/química , HumanosRESUMEN
Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.
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Mycobacterium tuberculosis , Tomografía de Emisión de Positrones , Trehalosa , Tuberculosis , Animales , Mycobacterium tuberculosis/metabolismo , Tomografía de Emisión de Positrones/métodos , Trehalosa/metabolismo , Tuberculosis/diagnóstico por imagen , Tuberculosis/microbiología , Tuberculosis/metabolismo , Humanos , Ratones , Radioisótopos de Flúor , Fluorodesoxiglucosa F18/metabolismo , Fluorodesoxiglucosa F18/química , Radiofármacos/metabolismo , Modelos Animales de Enfermedad , FemeninoRESUMEN
Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.
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Caspasa 2 , Inhibidores de Caspasas , Proteómica , Humanos , Caspasa 2/metabolismo , Caspasa 2/química , Proteómica/métodos , Inhibidores de Caspasas/farmacología , Inhibidores de Caspasas/química , Inhibidores de Caspasas/metabolismo , Estructura Molecular , Cisteína EndopeptidasasRESUMEN
Mapping the ligandability or potential druggability of all proteins in the human proteome is a central goal of mass spectrometry-based covalent chemoproteomics. Achieving this ambitious objective requires high throughput and high coverage sample preparation and liquid chromatography-tandem mass spectrometry analysis for hundreds to thousands of reactive compounds and chemical probes. Conducting chemoproteomic screens at this scale benefits from technical innovations that achieve increased sample throughput. Here we realize this vision by establishing the silane-based cleavable linkers for isotopically-labeled proteomics-tandem mass tag (sCIP-TMT) proteomic platform, which is distinguished by early sample pooling that increases sample preparation throughput. sCIP-TMT pairs a custom click-compatible sCIP capture reagent that is readily functionalized in high yield with commercially available TMT reagents. Synthesis and benchmarking of a 10-plex set of sCIP-TMT reveal a substantial decrease in sample preparation time together with high coverage and high accuracy quantification. By screening a focused set of four cysteine-reactive electrophiles, we demonstrate the utility of sCIP-TMT for chemoproteomic target hunting, identifying 789 total liganded cysteines. Distinguished by its compatibility with established enrichment and quantification protocols, we expect sCIP-TMT will readily translate to a wide range of covalent chemoproteomic applications.
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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.
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Cisteína , Proteínas , Cisteína/metabolismo , Proteínas/metabolismo , Péptidos/metabolismo , Orgánulos/metabolismo , Oxidación-ReducciónRESUMEN
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.
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Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.
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The plasma membrane proteome is a rich resource of functional 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 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.
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Mass spectrometry-based chemoproteomics has emerged as an enabling technology for functional biology and drug discovery. To address limitations of established chemoproteomics workflows, including cumbersome reagent synthesis and low throughput sample preparation, here, we established the silane-based cleavable isotopically labeled proteomics (sCIP) method. The sCIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)-protected amino acid building blocks, which enable the facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. sCIP is compatible with both MS1- and MS2-based quantitation, and the sCIP-MS2 method is distinguished by its click-assembled isobaric tags in which the reporter group is encoded in the sCIP capture reagent and balancer in the pan cysteine-reactive probe. The sCIP-MS2 workflow streamlines sample preparation with early stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost six-plex sample multiplexing. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.
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Cisteína , Silanos , Espectrometría de Masas , Indicadores y Reactivos , Proteómica/métodosRESUMEN
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.
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Cisteína , Proteómica , Animales , Ratones , Cisteína/metabolismo , Proteómica/métodos , Mitocondrias/metabolismo , Proteoma/metabolismo , Oxidación-ReducciónRESUMEN
Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.
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Cysteine chemoproteomics provides proteome-wide portraits of the ligandability or potential "druggability" for thousands of cysteine residues. Consequently, these studies are facilitating resources for closing the druggability gap, namely, achieving pharmacological manipulation of â¼96% of the human proteome that remains untargeted by U.S. Food and Drug Administration (FDA) approved small molecules. Recent interactive datasets have enabled users to interface more readily with cysteine chemoproteomics datasets. However, these resources remain limited to single studies and therefore do not provide a mechanism to perform cross-study analyses. Here we report CysDB as a curated community-wide repository of human cysteine chemoproteomics data derived from nine high-coverage studies. CysDB is publicly available at https://backuslab.shinyapps.io/cysdb/ and features measures of identification for 62,888 cysteines (24% of the cysteinome), as well as annotations of functionality, druggability, disease relevance, genetic variation, and structural features. Most importantly, we have designed CysDB to incorporate new datasets to further support the continued growth of the druggable cysteinome.
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Cisteína , Proteoma , Humanos , Cisteína/química , Proteoma/químicaRESUMEN
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. 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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Guest editors Keriann Backus, Zhengying Pan and Lyn Jones introduce the themed collection on Covalent Drug Discovery.
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Cysteine-directed chemoproteomic profiling methods yield high-throughput inventories of redox-sensitive and ligandable cysteine residues and therefore are enabling techniques for functional biology and drug discovery. However, the cumbersome nature of many sample preparation workflows, the requirements for large amounts of input material, and the modest yields of labeled peptides are limitations that hinder most chemoproteomics studies. Here, we report an optimized chemoproteomic 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 samples. We further tailor our SP3 method to specifically probe the redox proteome, which showcases the utility of the SP3 platform in multistep sample-preparation workflows. By implementing a customized workflow in the FragPipe computational pipeline, we achieve accurate MS1-based quantification, including for peptides containing multiple cysteine residues. Collectively these innovations enable enhanced high-throughput quantitative analysis of the cysteinome. This article includes detailed protocols for cysteine labeling with isotopically labeled iodoacetamide alkyne probes, biotinylation with CuAAC, sample cleanup with SP3, enrichment of cysteines with NeutrAvidin agarose beads, LC-FAIMS-MS/MS analysis, and FragPipe-IonQuant analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling of cysteines in human proteome and SP3-based sample cleanup Alternate Protocol 1: Labeling of cysteines in human proteome, SP3-based sample cleanup, and enrichment of cysteines for isoTOP-ABPP analysis Alternate Protocol 2: Labeling of cysteines in human proteome and SP3-based sample cleanup for redox proteome analysis Basic Protocol 2: Peptide-level cysteine enrichment Basic Protocol 3: LC-FAIMS-MS/MS analysis Basic Protocol 4: FragPipe data analysis.
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Proteoma , Proteómica , Proteínas Reguladoras de la Apoptosis , Cisteína/química , Humanos , Péptidos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
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.
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Química Clic , Cisteína , Alquinos/química , Azidas/química , Catálisis , Química Clic/métodos , Cobre/química , Reacción de Cicloadición , Cisteína/química , Humanos , Espectrometría de Masas en TándemRESUMEN
Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions. Although the resulting cysteine-azoline conjugate is stable, we highlight traceless decoupling of the conjugate to afford unmodified starting components in response to reducing conditions. We demonstrated that HAT probes reverse the reactivity of nucleophilic cysteine to electrophilic dehydroalanine (Dha) under mild basic conditions. We demonstrated the umpolung capability of HAT probes for the modification of cysteine on peptides and proteins with various nucleophiles. We demonstrated that HAT probes increase the mass sensitivity of the modified peptides and proteins by 100 fold as compared to the classical methods. Finally, we extended the application of HAT probes for specific modification of cysteines in a complex cell lysate mixture.
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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.
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Cisteína , Proteómica , Cisteína/química , Espectrometría de Masas/métodos , Oxidación-Reducción , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Proteome profiling by activated esters identified >9000 ligandable lysines but they are limited as covalent inhibitors due to poor hydrolytic stability. Here we report our efforts to design and discover a new series of tunable amine-reactive electrophiles (TAREs) for selective and robust labeling of lysine. The major challenges in developing selective probes for lysine are the high nucleophilicity of cysteines and poor hydrolytic stability. Our work circumvents these challenges by a unique design of the TAREs that form stable adducts with lysine and on reaction with cysteine generate another reactive electrophiles for lysine. We highlight that TAREs exhibit substantially high hydrolytic stability as compared to the activated esters and are non-cytotoxic thus have the potential to act as covalent ligands. We applied these alternative TAREs for the intracellular labeling of proteins in different cell lines, and for the selective identification of lysines in the human proteome on a global scale.
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LisinaRESUMEN
Nearly all FDA approved drugs and bioactive small molecules exert their effects by binding to and modulating proteins. Consequently, understanding how small molecules interact with proteins at an molecular level is a central challenge of modern chemical biology and drug development. Complementary to structure-guided approaches, chemoproteomics has emerged as a method capable of high-throughput identification of proteins covalently bound by small molecules. To profile noncovalent interactions, established chemoproteomic workflows typically incorporate photoreactive moieties into small molecule probes, which enable trapping of small molecule-protein interactions (SMPIs). This strategy, termed photoaffinity labelling (PAL), has been utilized to profile an array of small molecule interactions, including for drugs, lipids, metabolites, and cofactors. Herein we describe the discovery of photocrosslinking chemistries, including a comparison of the strengths and limitations of implementation of each chemotype in chemoproteomic workflows. In addition, we highlight key examples where photoaffinity labelling has enabled target deconvolution and interaction site mapping.