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

• Published in: Analytical chemistry (Washington) Vol. 92; no. 7; pp. 4742 - 4748
• Main Authors: Li, Qingqing, Zhang, Yang, Huang, Jingnan, Wu, Zhen, Tang, Langlang, Huang, Lin, Zhang, Xumin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.04.2020
• Subjects: Cation exchange; Cation exchanging; Cations - chemistry; Chemistry; Chromatography; Chromatography, Ion Exchange; Digestion; Esterification; HEK293 Cells; HeLa Cells; Humans; Mass spectrometry; Mass spectroscopy; Metalloproteases - analysis; Metalloproteases - metabolism; Parameter identification; Protein C; Proteins; Proteomes; Tomatoes
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b05280

Decoding protein C-termini is a challenging task in protein chemistry using conventional chemical and 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 progress is being 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 the BaSCX method, for basic strong cation exchange chromatography, for C-terminome studies. Taking advantage of carboxylic amidation, LysargiNase digestion, and optimized search parameters, BaSCX enables identification of 1806 and 1812 database-annotated human protein C-termini from HeLa and 293T cells, resepctively, by triplicate experiments using 40 μg proteins each. Combined together, 2151 human protein C-termini, nearly three times the recently reported largest human C-terminome data set, are reported in this study. Similar results were acquired in different organisms, including mice, C. elegans, and tomatoes. Furthermore, we report for the first time the discovery of C-terminal-specific modifications using a proteomic approach, including three methyl-esterified protein C-termini and 16 α-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.