Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL) to Identify Binding Sites of Small Molecules on a Proteome-Wide Scale.
Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL) to Identify Binding Sites of Small Molecules on a Proteome-Wide Scale.
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DOI：
10.1002/cpch.75
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发表时间：
2019-12-01
影响因子：
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通讯作者：
Woo, Christina M
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文献类型：
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作者：
Flaxman, Hope A;Miyamoto, David K;Woo, Christina M
鉴定和表征小分子与蛋白质的相互作用是理解生物活性小分子作用机制的关键。光亲和标记法(PAL)能够捕获非共价相互作用，以便用质谱仪(MS)进行浓缩和无偏分析。富含蛋白质组的定量蛋白质组学揭示了潜在的相互作用，结合位点的MS表征提供了对相互作用的验证和结构洞察。在这里，我们描述了利用PAL的小分子相互作用组图(SIM-PAL)来识别小分子的蛋白质靶标和结合位置。细胞暴露在二氮杂炔功能化的小分子中，结合作用在紫外线照射下被共价捕获。一个同位素编码的、可酸分解的生物素叠氮化物手柄通过铜催化的叠氮-炔环加成反应连接到结合蛋白上。生物素标记的蛋白质被浓缩，用于珠状消化和定量蛋白质组学。手柄的酸裂解释放珠结合的结合肽，用于MS分析和结合位点的同位素定向分配。基本协议1：处理活细胞后生成小分子结合蛋白样本替代协议：细胞裂解液处理后生成小分子结合蛋白样本基本协议2：铜催化叠氮-炔环加成功能化和标记多肽的浓缩支持协议1：合成酸可切割、同位素编码的生物素吡啶叠氮句柄支持协议2：通过免疫印迹监测浓缩基本协议3：质谱分析以确定相互作用的蛋白质和结合位点。
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.