Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry
Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry
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DOI：
10.1074/mcp.tir118.001099
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发表时间：
2019-05-01
影响因子：
7
通讯作者：
O'Donoghue, Anthony J.
中科院分区：
文献类型：
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作者：
Lapek, John D., Jr.;Jiang, Zhenze;O'Donoghue, Anthony J.
蛋白质水解是生命不可分割的组成部分，与许多疾病过程有关。为了提高我们对肽酶功能的理解，必须开发工具来揭示底物特异性和裂解效率。在这里，我们将串联质量标签（TMTs）的定量能力与已建立的肽裂解测定相结合，产生定量的多重底物质谱分析（qMSP-MS）。该实验用木瓜蛋白酶（一种特性良好的半胱氨酸肽酶）进行了验证，得到了275个独特肽键平行水解的裂解效率值。为了证明该实验的可行性，我们发现qMSP-MS可以揭示膜内方形肽酶的底物特异性，以及定义复杂生物样品（包括肺癌细胞系的分泌物）中的数百种蛋白水解活性。重要的是，我们的qMSP-MS库使用的是末端未修饰的合成肽，这使我们不仅可以表征内肽酶活性，还可以表征外肽酶活性。每个被切割的肽序列可按周转率排序，最佳底物的氨基酸序列可用于设计荧光报告底物。发现被在疾病部位活跃的肽酶选择性切割的肽底物，突出了qMSP-MS在指导癌症和传染病的肽酶激活药物开发方面的潜力。生物信息学学报（自然科学版），2016；DOI: 10.1074/ mcp。TIR118.001099。
Proteolysis is an integral component of life and has been implicated in many disease processes. To improve our understanding of peptidase function, it is imperative to develop tools to uncover substrate specificity and cleavage efficiency. Here, we combine the quantitative power of tandem mass tags (TMTs) with an established peptide cleavage assay to yield quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS). This assay was validated with papain, a well-characterized cysteine peptidase, to generate cleavage efficiency values for hydrolysis of 275 unique peptide bonds in parallel. To demonstrate the breath of this assay, we show that qMSP-MS can uncover the substrate specificity of minimally characterized intramembrane rhomboid peptidases, as well as define hundreds of proteolytic activities in complex biological samples, including secretions from lung cancer cell lines. Importantly, our qMSP-MS library uses synthetic peptides whose termini are unmodified, allowing us to characterize not only endo-but also exo-peptidase activity. Each cleaved peptide sequence can be ranked by turnover rate, and the amino acid sequence of the best substrates can be used for designing fluorescent reporter substrates. Discovery of peptide substrates that are selectively cleaved by peptidases which are active at the site of disease highlights the potential for qMSP-MS to guide the development of peptidase-activating drugs for cancer and infectious disease. Molecular & Cellular Proteomics 18: 968-981, 2019. DOI: 10.1074/ mcp. TIR118.001099.