A tandem orthogonal proteolysis strategy for high-content chemical proteomics
A tandem orthogonal proteolysis strategy for high-content chemical proteomics
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DOI：
10.1021/ja0532842
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发表时间：
2005-07-20
影响因子：
15
通讯作者：
Cravatt, BF
中科院分区：
文献类型：
--
作者：
Speers, AE;Cravatt, BF
蛋白质组学领域的目标是为真核和原核生物基因组编码的众多蛋白质产物分配功能。为此，化学策略已经成为一种强大的手段，可以基于共同的功能特性来丰富特定类别的蛋白质，例如催化活性[基于活性的蛋白质图谱(ABPP)]和翻译后修饰状态。化学蛋白质组学实验中的理论信息量大大超过了实际获得的数据，这在很大程度上是由于现有分析技术的局限性。在这里，我们提出了一种用于高含量化学蛋白质组学的串联正交蛋白质分解(TOP)策略，该策略能够并行地表征探针标记的蛋白质和探针修饰的位置。TOP方法利用“点击化学”在探针标记的蛋白质上引入一个多功能标签，该标签既包含用于浓缩蛋白质的生物素基团，也包含用于选择性释放探针修饰的多肽的烟草蚀刻病毒(TEV)蛋白酶裂解位点。在链霉亲和素珠上捕获后，通过两步蛋白分解策略(分别为胰酶和TEV)顺序鉴定探针的蛋白质靶标及其标记位置。我们应用TOP方法来表征组织蛋白质组中的磺酸酯ABPP探针的靶标，从而发现了许多活性位点标记的酶。还鉴定了调节位点上修饰的酶和功能未知的蛋白质。这些发现表明，磺酸酯探针针对的是广泛的功能残基，并突出了基于TOP的化学蛋白质组学在表征蛋白质和调节其活性的残基方面的价值。
The field of proteomics aims to assign functions to the numerous protein products encoded by eukaryotic and prokaryotic genomes. Toward this end, chemical strategies have emerged as a powerful means to enrich specific classes of proteins based on shared functional properties, such as catalytic activity [activity-based protein profiling (ABPP)], and post-translational modification state. The theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured, due in large part to limitations in existing analytical technologies. Here, we present a tandem orthogonal proteolysis (TOP) strategy for high-content chemical proteomics that enables the parallel characterization of probe-labeled proteins and sites of probe modification. The TOP approach exploits “click chemistry” to introduce a multifunctional tag onto probe-labeled proteins that contains both a biotin group for protein enrichment and a tobacco etch virus (TEV) protease cleavage site for selective release of probe-modified peptides. Following capture on streptavidin beads, protein targets of probes and their sites of labeling are sequentially identified by a two-step proteolysis strategy (trypsin and TEV, respectively). We apply the TOP method to characterize targets of sulfonate ester ABPP probes in tissue proteomes, resulting in the discovery of numerous active site-labeled enzymes. Enzymes modified on regulatory sites and proteins of unknown function were also identified. These findings indicate that a wide range of functional residues are targeted by sulfonate ester probes and highlight the value of TOP-based chemical proteomics for the characterization of proteins and the residues that regulate their activity.