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 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.

采用传统化学与酶学方法解析蛋白质C端，是蛋白质化学领域一项极具挑战性的工作。随着现代质谱仪的快速发展，诸多基于质谱（MS）的先进蛋白质C端分析方法相继被建立。尽管该领域不断取得显著进展，但仍需开发更高效的方法，以实现蛋白质组规模的蛋白质C端（C端组）全景解析，进而助力阐明其生物学功能。本研究报道了一种用于C端组研究的碱性强阳离子交换色谱（BaSCX）法。该方法借助羧酸酰胺化、LysargiNase酶解以及优化的检索参数，通过每份样品40微克蛋白的三次重复实验，分别从HeLa细胞与293T细胞中鉴定出1806和1812个数据库注释的人类蛋白质C端。本研究共鉴定得到2151个人类蛋白质C端，约为目前已报道的最大规模人类C端组数据集的三倍。在小鼠、秀丽隐杆线虫（C. elegans）与番茄等不同物种中也获得了类似实验结果。此外，本研究首次利用蛋白质组学方法发现了C端特异性修饰，包括3个甲基酯化修饰的蛋白质C端与16个α酰胺化修饰的蛋白质C端，充分证明了BaSCX法在C端组学研究中的优异性能与巨大应用潜力。相关数据集可通过蛋白质组交换库（ProteomeXchange）获取，登录号为PXD016317。