Manitoba Centre for Proteomics and Systems Biology, Winnipeg, MB, Canada.
Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada.
Methods Mol Biol. 2023;2718:99-110. doi: 10.1007/978-1-0716-3457-8_6.
Many proteolytic cleavage events cannot be covered with conventional trypsin-based N-terminomics workflows. These typically involve the derivatization of protein N-termini and Lys residues as an initial step, such that trypsin will cleave C-terminal of arginine but not lysine residues (ArgC-like cleavage). From 20,422 reviewed human protein sequences in Uniprot, 3597 have known N-terminal signal peptides. An in silico ArgC-like digestion of the corresponding 3597 mature protein sequences reveals that-even for these well-known and well-studied proteolytic events-trypsin-based N-terminomics workflows may miss up to 50% of signaling cleavage events as the corresponding neo-N-terminal peptides will have an unfavorable length of <7 (875 peptides) or >30 (911 peptides) amino acids. In this chapter, we provide a protocol that can be applied to all kinds of samples to improve access to this "inaccessible" N-terminome, by making use of the alternative, broad-specificity protease subtilisin for fast and reproducible digestion of proteins.
许多蛋白水解切割事件无法通过传统的基于胰蛋白酶的 N 端组学工作流程来覆盖。这些通常涉及蛋白质 N 末端和赖氨酸残基的衍生化作为初始步骤，使得胰蛋白酶将在精氨酸的 C 末端而不是赖氨酸残基处切割（ArgC 样切割）。在 Uniprot 中审查的 20422 个人类蛋白质序列中，有 3597 个具有已知的 N 端信号肽。对相应的 3597 个成熟蛋白质序列进行计算机模拟的 ArgC 样消化表明-即使对于这些众所周知和研究充分的蛋白水解事件-基于胰蛋白酶的 N 端组学工作流程可能会错过多达 50%的信号切割事件，因为相应的新 N 端肽的长度不合适，<7（875 个肽）或>30（911 个肽）个氨基酸。在本章中，我们提供了一种可以应用于各种样品的方案，通过利用替代的、广谱特异性蛋白酶枯草杆菌蛋白酶来快速且可重复地消化蛋白质，从而可以更好地获得这种“不可及”的 N 端组。
