Spatially Resolved Tagging of Proteolytic Neo-N termini with Subtiligase-TM

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.