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Basic Strong Cation Exchange Chromatography, BaSCX, a Highly Efficient Approach for C-Terminomic Studies Using LysargiNase Digestion.

Decoding protein C-termini is a challenging task in protein chemistry using conventional chemical/enzymatic approaches. With the rapid development in modern mass spectrometer, many advanced mass spectrometry (MS) based protein C-termini analysis approaches have been established. Although great progresses have been continually achieved, it is still necessary to develop more efficient approaches in order to discover a proteome-scale protein C-termini (C-terminome), and consequently to help understand their biological functions. In this report, we describe a simple method, termed BaSCX, for basic strong cation exchange chromatography, to study C-terminome. Taking advantage of C-term carboxylic protection, LysargiNase digestion and optimized search parameters, BaSCX enables to identify 1806 and 1812 database annotated human protein C-termini from HeLa and 293T cell by triplicate experiments using 40 μg proteins each. Combined together, 2151 human protein C-termini, nearly three times of the recently reported largest human C-terminome dataset, are reported in this study. Similar results were acquired in different organisms, including mouse, C. elegans and tomato. Furthermore, we report for the first time the discovery of C-terminal specific modifications using proteomic approach, including three methyl-esterified protein C-termini and 16 -amidated protein C-termini, demonstrating the excellent performance and great potential of BaSCX in C-terminomic studies. Data are available via ProteomeXchange with identifier PXD016317.