Discovery of Electrophiles and Profiling of Enzyme Cofactors.
Discovery of Electrophiles and Profiling of Enzyme Cofactors.
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发现电物质和酶辅因子的分析。
DOI：
10.1002/cpch.86
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发表时间：
2020-12
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基于反极性活性的蛋白质谱分析（RP‐ABPP）是一种化学蛋白质组学方法，该方法使用亲核探针进行“点击”化学，部署到培养的活细胞中，以捕获、免疫沉淀和鉴定蛋白质结合的亲电体。RP-ABPP用于表征反应性亲电翻译后修饰（PTM）及其蛋白质的结构和功能，这可能会发现未知或新的功能。RP-ABPP已被证明是一种多功能方法，可用于监测亲电辅因子的代谢调节，使用S-腺苷-L-甲硫氨酸脱羧酶（AMD 1）中的甘氨酰辅因子，并发现人类细胞中蛋白质的新型亲电修饰，如secernin-3（SCRN 3）上的甘氨酰修饰。这些辅因子不能通过序列预测，因此该区域相对不发达。RP-ABPP是发现这种亲电体的唯一全球性、无偏见的方法。在这里，我们描述了这些实验的效用，并提供了一个详细的协议从头发现，定量和全球分析的亲电功能的蛋白质。版权所有2020作者。 基本方案1：探针反应蛋白的鉴定和定量基本方案2：探针标记位点的表征基本方案3：亲电体结构的测定和定量
Reverse‐polarity activity‐based protein profiling (RP‐ABPP) is a chemical proteomics approach that uses nucleophilic probes amenable to “click” chemistry deployed into living cells in culture to capture, immunoprecipitate, and identify protein‐bound electrophiles. RP‐ABPP is used to characterize the structure and function of reactive electrophilic post‐translational modifications (PTMs) and the proteins harboring them, which may uncover unknown or novel functions. RP‐ABPP has demonstrated utility as a versatile method to monitor the metabolic regulation of electrophilic cofactors, using a pyruvoyl cofactor in S‐adenosyl‐l‐methionine decarboxylase (AMD1), and to discover novel types of electrophilic modifications on proteins in human cells, such as the glyoxylyl modification on secernin‐3 (SCRN3). These cofactors cannot be predicted by sequence, and therefore this area is relatively undeveloped. RP‐ABPP is the only global, unbiased approach to discover such electrophiles. Here, we describe the utility of these experiments and provide a detailed protocol for de novo discovery, quantitation, and global profiling of electrophilic functionality of proteins. © 2020 The Authors. Basic Protocol 1: Identification and quantification of probe‐reactive proteins Basic Protocol 2: Characterization of the site of probe labeling Basic Protocol 3: Determination and quantitation of electrophile structure