Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry
Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry
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DOI：
10.1016/j.ijms.2006.09.020
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发表时间：
2007-01-01
影响因子：
1.8
通讯作者：
Hendrickson, Ronald C.
中科院分区：
文献类型：
--
作者：
Yates, Nathan A.;Deyanova, Ekaterina G.;Hendrickson, Ronald C.
大约 2% 的人类基因组编码蛋白酶。然而不幸的是，大多数这些酶的生物学作用仍然不清楚，因为生理底物通常是未知的，并且很难使用传统方法进行识别。我们开发了一种基于 FTMS 分析和差示质谱 (dMS) 的蛋白质组学实验，以使用分级的人血浆作为候选底物池来识别蛋白酶的候选内源底物。在此，我们报告了用于鉴定抗肽酶 P2 (APP2) 和二肽基肽酶 4 (DPP-4)（一种对治疗 2 型糖尿病具有治疗意义的肽酶）体外底物的概念验证实验。对于这两种蛋白酶，先前验证的添加到人血浆池中的肽底物被鉴定出来。值得注意的是，差示质谱实验还鉴定了人血浆亚组分中每种肽酶的新底物。对复杂的人血浆池中的这些肽进行针对性的 MS/MS 分析并手动确认氨基酸序列，从而鉴定出这些底物。化学合成了新型 DPP-4 底物 EPLGRQLTSGP，并通过体外 DPP-4 酶测定测定了裂解动力学。 DPP-4 介导的切割的表观二阶速率常数 (k(cat)/K-M) 确定为 2.3 x 10(5) M-1 s(-1)，证实该肽在体外被肽酶有效加工。总的来说，这些结果表明差示质谱法有可能从人血浆池中识别目标蛋白酶的候选内源底物。重要的是，对内源底物的了解可以提供对这些酶的生物学的有用见解，并为监测其体内活性提供有用的生物标志物。 (c) 2006 Elsevier B.V. 保留所有权利。
Approximately 2% of the human genome encodes for proteases. Unfortunately, however, the biological roles of most of these enzymes remain poorly defined, since the physiological substrates are typically unknown and are difficult to identify using traditional methods. We have developed a proteomics experiment based on FTMS profiling and differential mass spectrometry (dMS) to identify candidate endogenous substrates of proteases using fractionated human plasma as the candidate substrate pool. Here we report proof-of-concept experiments for identifying in vitro substrates of antinopeptidase P2, (APP2) and dipeptidyl peptidase 4 (DPP-4), a peptidase of therapeutic interest for the treatment of type 2 diabetes. For both proteases, previously validated peptide substrates spiked into the human plasma pool were identified. Of note, the differential mass spectrometry experiments also identified novel substrates for each peptidase in the subfraction of human plasma. Targeted MS/MS analysis of these peptides in the complex human plasma pool and manual confirmation of the amino acid sequences led to the identification of these substrates. The novel DPP-4 substrate EPLGRQLTSGP was chemically synthesized and cleavage kinetics were determined in an in vitro DPP-4 enzyme assay. The apparent second order rate constant (k(cat)/K-M) for DPP-4-mediated cleavage was determined to be 2.3 x 10(5) M-1 s(-1) confirming that this peptide is efficiently processed by the peptidase in vitro. Collectively, these results demonstrate that differential mass spectrometry has the potential to identify candidate endogenous substrates of target proteases from a human plasma pool. Importantly, knowledge of the endogenous substrates can provide useful insight into the biology of these enzymes and provides useful biomarkers for monitoring their activity in vivo. (c) 2006 Elsevier B.V. All rights reserved.