Fan Chao, Shi Zhaomei, Pan Yiting, Song Zifeng, Zhang Wanjun, Zhao Xinyuan, Tian Fang, Peng Bo, Qin Weijie, Cai Yun, Qian Xiaohong
National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine , Beijing 102206, China.
Anal Chem. 2014 Feb 4;86(3):1452-8. doi: 10.1021/ac402696b. Epub 2014 Jan 21.
In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage.
在全基因组分析时代，基于质谱的自下而上策略现在被认为是深入蛋白质组学分析最强大的方法。作为该策略的一部分，将蛋白质高效且完全地酶解为肽段对于成功进行具有深度覆盖的蛋白质组分析至关重要。为实现这一目标，人们采用了延长酶解时间和使用多种蛋白酶的方法。所需的长时间酶解和繁琐的样品处理步骤严重限制了样品处理通量。尽管使用固定化蛋白酶大大缩短了酶解时间，但对极其复杂的蛋白质组样品进行高效蛋白水解仍然是一项具有挑战性的任务。在此，我们提出一种基于双基质的互补酶解方法，该方法使用两种通过将胰蛋白酶附着在疏水或亲水聚合物刷修饰的纳米颗粒上制备的具有相反基质疏水性的固定化胰蛋白酶。纳米颗粒上的聚合物刷作为大量胰蛋白酶固定化的三维支撑体，可实现超快且高效的蛋白质酶解。更重要的是，这两种固定化胰蛋白酶在蛋白质酶解方面表现出高度互补性，对于酵母和小鼠肝脏膜蛋白，肽段鉴定的重叠率仅约为60%。将这两种固定化胰蛋白酶一起应用进行互补酶解可显著增强蛋白质和肽段的鉴定。此外，基于双基质的互补酶解在膜蛋白酶解方面表现出特别的优势，与使用游离蛋白酶的溶液酶解相比，鉴定出的肽段数量增加了一倍，证明了其作为一种有前途的替代方法以促进具有更高蛋白质序列覆盖率的蛋白质组分析的潜力。
