LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome
LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome
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基于 LysargiNase 和化学衍生化的策略，促进 C 端组的深入分析
DOI：
10.1021/acs.analchem.9b03543
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发表时间：
2019-11-19
影响因子：
7.4
通讯作者：
Tan, Minjia
中科院分区：
文献类型：
--
作者：
Hu, Hao;Zhao, Wensi;Tan, Minjia
由于传统鸟枪法蛋白质组学中蛋白质 C 末端的丰度较低，因此对蛋白质 C 末端进行全面鉴定非常具有挑战性。已经开发了几种富集策略来促进 C 末端肽的检测。先前方法的一个主要问题是 C 末端覆盖范围有限。在此，我们将 LysargiNase 消化、新 N 末端化学乙酰化和 a 离子辅助肽匹配整合到基于聚（烯丙胺）的 C 末端组学（称为 LAACTer）中。在该策略中，我们利用 LysargiNase（一种对 Lys 和 Arg 残基 N 端具有裂解特异性的蛋白酶）来覆盖以前无法识别的 C 端，并采用化学乙酰化和 a 离子辅助肽匹配来有效促进肽鉴定。 LAACTer 的三次重复从 293T 细胞的蛋白质组中鉴定出总共 834 个 C 末端，与使用原始工作流程的平行实验相比，覆盖范围扩大了 164%（多了 643 个独特的 C 末端）。与最大的人类 C 末端数据集（包含 800-900 个 C 末端）相比，LAACTer 不仅达到了相当的分析深度，而且还产生了 465 个先前未识别的 C 末端。在一项基于 SILAC（细胞培养物中氨基酸稳定同位素标记）的定量研究中，用于鉴定 GluC 裂解产物，LAACTer 定量的 C 端肽比原始工作流程多了 300%。使用LAACTer和原始工作流程，我们对293T细胞的C端序列进行了全局分析。原始的和加工后的 C 末端显示出不同的序列模式，这意味着调节蛋白质稳定性的“C 末端规则”可能比氨基酸基序更复杂。总之，我们认为 LAACTer 可能是深入 C 末端组学的强大蛋白质组学工具，并将有利于更好地了解蛋白质 C 末端的功能。
Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.