Terminomics for plant protease substrate discovery: successes, challenges, and new approaches

Plants contain hundreds of proteases that are involved in the regulation of virtually all cellular processes. Some proteases act as molecular shredders, resulting in degradation of their substrates. Others act more like scissors, cutting substrate proteins in a limited manner at specific sites to alter their activity, location, and function. Such tailored proteoforms share their sequence with the precursor form and sometimes only differ by the new, proteolytically modified polypeptide termini. Identification of protein termini is mandatory for unambiguous identification, but challenging in standard MS-based proteomics. Over the last two decades, various methods for the enrichment of N- and C-terminal peptides have been developed to enable proteome-wide characterization. Here we briefly introduce major approaches to protein termini enrichment and review current applications for plant protease substrate identification and profiling of proteolytic cleavage events in vivo. We highlight both successes and limitations, and discuss current improvements in sample preparation, data acquisition, MS instrumentation, and data analysis that promise to increase sensitivity, robustness, and ultimately utility of termini-centric proteomics.