Wang Heping, Jiao Fenglong, Gao Fangyuan, Zhao Xinyuan, Zhao Yan, Shen Yehua, Zhang Yangjun, Qian Xiaohong
Key Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi'an, Shaanxi, 710069, China.
State Key Laboratory of Proteomics, National Center for Protein Science Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
Anal Bioanal Chem. 2017 Mar;409(8):2179-2187. doi: 10.1007/s00216-016-0163-z. Epub 2017 Jan 11.
Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 μg mg). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis. Graphical abstract Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization.
深度且高效的蛋白质水解是基于质谱的自下而上蛋白质组学的关键前提。传统的溶液内消化很难满足要求，除非延长消化时间并增加酶用量，而这会使整个工作流程既耗时又昂贵。通过将多种蛋白酶固定在固体载体上，可以有效改善上述问题。在这项工作中，设计并制备了共价有机框架包覆的磁性石墨烯（MG@TpPa-1）作为一种新型酶载体，用于以高负载量（高达268μg mg）共价固定胰蛋白酶。得益于磁性石墨烯和共价有机框架的优势，这种新型胰蛋白酶生物反应器成功应用于模型蛋白质的酶解，其消化效率、稳定性和可重复使用性都有显著提高。在短短2分钟内即可实现完全消化。对于从长柄扁桃中提取的蛋白质进行消化时，共鉴定出2833个蛋白质组，略多于通过12小时溶液内消化获得的蛋白质组（2739个蛋白质组）。所有结果表明，MG@TpPa-（1）胰蛋白酶是高通量蛋白质组学分析中样品制备的极佳候选物。图形摘要 制备了共价有机框架包覆的磁性石墨烯作为高效固定胰蛋白酶的新型载体。
