Profiling serine hydrolase activities in complex proteomes
Profiling serine hydrolase activities in complex proteomes
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DOI：
10.1021/bi002579j
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发表时间：
2001-04-03
期刊：
影响因子：
2.9
通讯作者：
Cravatt, BF
中科院分区：
文献类型：
--
作者：
Kidd, D;Liu, YS;Cravatt, BF
丝氨酸水解酶代表高等真核生物中最大且最多样化的酶家族之一，包括多种蛋白酶、脂肪酶、酯酶和酰胺酶。许多丝氨酸水解酶的活性受到翻译后机制的严格调控，限制了标准基因组学和蛋白质组学方法对这些酶的功能表征的适用性。为了促进复杂蛋白质组中丝氨酸水解酶活性的整体分析，最近合成了生物素化的氟磷酸盐（FP-生物素），并被证明可以作为该酶家族几个成员的基于活性的探针。然而，FP-生物素与给定蛋白质组中存在的全部活性丝氨酸水解酶的反应程度在很大程度上仍未被探索。在此，我们描述了 FP-生物素变体的合成和用途，其中该试剂的疏水性烷基链连接基被更亲水性的聚（乙二醇）部分（FP-peg-生物素）取代。当与可溶性蛋白质组和膜蛋白质组一起孵育延长反应时间时，FP-生物素和 FP-peg-生物素产生相似的“最大覆盖”丝氨酸水解酶活性谱。然而，动力学分析表明，几种丝氨酸水解酶与每种 FP 试剂的反应速率不同。在使用生物素化 FP 直接检查复杂蛋白质组中可逆丝氨酸水解酶抑制剂的靶选择性的研究中，利用了这些速率差异。最后，开发了一种基于亲和素的 FP 生物素化蛋白亲和分离的通用方法，允许快速、同时鉴定多种丝氨酸肽酶、脂肪酶和酯酶。总的来说，这些研究表明，生物素化 FP 等化学探针可以极大地加速复杂蛋白质组中活性酶的功能表征和分子鉴定。
Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, Lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.