Flaxman Hope A, Miyamoto David K, Woo Christina M
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts.
Curr Protoc Chem Biol. 2019 Dec;11(4):e75. doi: 10.1002/cpch.75.
Identification and characterization of small molecule-protein interactions is critical to understanding the mechanism of action of bioactive small molecules. Photo-affinity labeling (PAL) enables the capture of noncovalent interactions for enrichment and unbiased analysis by mass spectrometry (MS). Quantitative proteomics of the enriched proteome reveals potential interactions, and MS characterization of binding sites provides validation and structural insight into the interactions. Here, we describe the identification of the protein targets and binding sites of a small molecule using small molecule interactome mapping by PAL (SIM-PAL). Cells are exposed to a diazirine-alkyne-functionalized small molecule, and binding interactions are covalently captured upon UV irradiation. An isotopically coded, acid-cleavable biotin azide handle is attached to the conjugated proteins using copper-catalyzed azide-alkyne cycloaddition. Biotin-labeled proteins are enriched for on-bead digestion and quantitative proteomics. Acid cleavage of the handle releases the bead-bound conjugated peptides for MS analysis and isotope-directed assignment of the binding site. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a small molecule-conjugated protein sample following treatment of live cells Alternate Protocol: Generation of a small molecule-conjugated protein sample following treatment of cell lysate Basic Protocol 2: Copper-catalyzed azide-alkyne cycloaddition functionalization and enrichment of labeled peptides Support Protocol 1: Synthesis of acid-cleavable, isotopically coded biotin picolyl azide handle Support Protocol 2: Monitoring enrichment by immunoblotting Basic Protocol 3: Mass spectrometry analysis to identify interacting proteins and conjugation sites.
鉴定和表征小分子与蛋白质的相互作用对于理解生物活性小分子的作用机制至关重要。光亲和标记（PAL）能够捕获非共价相互作用，以便通过质谱（MS）进行富集和无偏分析。对富集蛋白质组进行定量蛋白质组学分析可揭示潜在的相互作用，而结合位点的MS表征则为相互作用提供验证和结构洞察。在此，我们描述了使用光亲和标记小分子相互作用组图谱（SIM-PAL）鉴定小分子的蛋白质靶点和结合位点的方法。将细胞暴露于重氮丙啶-炔烃功能化的小分子中，经紫外线照射后共价捕获结合相互作用。使用铜催化的叠氮化物-炔烃环加成反应，将同位素编码的、酸可裂解的生物素叠氮化物手柄连接到共轭蛋白质上。对生物素标记的蛋白质进行富集，用于珠上消化和定量蛋白质组学分析。手柄的酸裂解释放出与珠结合的共轭肽，用于MS分析和结合位点的同位素导向定位。© 2019 John Wiley & Sons, Inc. 基本方案1：活细胞处理后生成小分子共轭蛋白质样品 替代方案：细胞裂解液处理后生成小分子共轭蛋白质样品 基本方案2：铜催化的叠氮化物-炔烃环加成功能化及标记肽的富集 支持方案1：酸可裂解的、同位素编码的生物素吡啶甲基叠氮化物手柄的合成 支持方案2：通过免疫印迹监测富集情况 基本方案3：质谱分析以鉴定相互作用的蛋白质和共轭位点。
