Pan Yanbo, Cheng Kai, Mao Jiawei, Liu Fangjie, Liu Jing, Ye Mingliang, Zou Hanfa
Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
Anal Bioanal Chem. 2014 Oct;406(25):6247-56. doi: 10.1007/s00216-014-8071-6. Epub 2014 Aug 19.
Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.
在鸟枪法蛋白质组学中，胰蛋白酶是一种常用的将蛋白质消化成肽段的蛋白酶，但很少有研究尝试系统地研究胰蛋白酶催化蛋白质组样品中蛋白质消化的动力学。在本研究中，我们通过三重稳定同位素二甲基标记应用定量蛋白质组学来研究胰蛋白酶催化裂解的动力学。结果发现，胰蛋白酶切割赖氨酸（K）和精氨酸（R）残基的C末端，对R的切割速率更高。被中性残基包围的切割位点可以快速切割，而那些带有相邻带电残基（D/E/K/R）或脯氨酸残基（P）的切割位点则切割较慢。在蛋白质组样品中，大量具有不同物理化学性质的蛋白质共存。如果任何一种类型的蛋白质都能被优先消化，那么可以采用有限消化来降低样品复杂性。然而，我们发现蛋白质丰度和其他物理化学性质，如分子量（Mw）、亲水性总平均值（GRAVY）、脂肪族指数和等电点（pI）与蛋白质的消化优先级没有显著相关性。
