The Journal of Membrane Biology ( IF 2.3 ) Pub Date : 2021-02-18 , DOI: 10.1007/s00232-021-00171-4 Amy M Weeks 1
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Mass spectrometry-based proteomics has been used successfully to identify substrates for proteases. Identification of protease substrates at the cell surface, however, can be challenging since cleavages are less abundant compared to other cellular events. Precise methods are required to delineate cleavage events that take place in these compartmentalized areas. This article by up-and-coming scientist Dr. Amy Weeks, an Assistant Professor at the University of Wisconsin-Madison, provides an overview of methods developed to identify protease substrates and their cleavage sites at the membrane. An overview is presented with the pros and cons for each method and in particular the N-terminomics subtiligase-TM method, developed by Dr. Weeks as a postdoctoral fellow in the lab of Dr. Jim Wells at University of California, San Francisco, is described in detail. Subtiligase-TM is a genetically engineered subtilisin protease variant that acts to biotinylate newly generated N termini, hence revealing new cleavage events in the presence of a specific enzyme, and furthermore can precisely identify the cleavage P1 site. Importantly, this proteomics method is compatible with living cells. This method will open the door to understanding protein shedding events at the biological membrane controlled by proteases to regulate biological processes.
Graphic Abstract
中文翻译：
用 Subtiligase-TM 对蛋白水解 Neo-N 末端进行空间分辨标记
基于质谱的蛋白质组学已成功用于鉴定蛋白酶的底物。然而，在细胞表面鉴定蛋白酶底物可能具有挑战性，因为与其他细胞事件相比，裂解较少。需要精确的方法来描述在这些分隔区域中发生的切割事件。这篇由威斯康星大学麦迪逊分校助理教授、崭露头角的科学家 Amy Weeks 博士撰写的文章概述了为识别蛋白酶底物及其在膜上的切割位点而开发的方法。概述了每种方法的优缺点，特别是 N-terminomics subtiligase-TM 方法，该方法由 Weeks 博士作为加州大学旧金山分校 Jim Wells 博士实验室的博士后研究员开发，有详细描述。Subtiligase-TM 是一种基因工程枯草杆菌蛋白酶变体，其作用是对新产生的 N 末端进行生物素化，从而在特定酶存在的情况下揭示新的切割事件，并且可以精确识别切割 P1 位点。重要的是，这种蛋白质组学方法与活细胞兼容。这种方法将为了解蛋白酶控制的生物膜上的蛋白质脱落事件打开大门，以调节生物过程。
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