Moore Stephanie, Hess Stephanie, Jorgenson James
Chemistry Department, University of North Carolina at Chapel Hill, NC 27599, United States.
Chemistry Department, University of North Carolina at Chapel Hill, NC 27599, United States.
J Chromatogr A. 2016 Dec 9;1476:1-8. doi: 10.1016/j.chroma.2016.11.021. Epub 2016 Nov 15.
Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10s volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein's molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15h.
尽管液相色谱和质谱检测技术发展得更快，但蛋白质组学分析中标准的溶液内蛋白质消化方法相对仍未改变。典型的溶液内胰蛋白酶蛋白质消化通常是工作流程中最慢的部分，尽管它是最重要的部分之一。开发一种具有快速性能的用于在线蛋白质消化的高效固定化酶反应器（IMER）将大大减少蛋白质组学工作流程中的分析时间。本文介绍了一种用于在线蛋白质消化的基于二氧化硅的IMER的开发，该反应器在有机流动相存在的情况下，对模型蛋白质和由酵母细胞裂解物不溶部分组成的复杂样品都能实现快速消化。在IMER和溶液内消化之间评估的蛋白质序列覆盖率和鉴定结果具有可比性。总体而言，对于在柱上仅具有10秒体积停留时间的酵母细胞裂解物，IMER鉴定出507种蛋白质，而溶液内消化鉴定出490种。两种消化方法之间基于鉴定出的蛋白质的分子量或等电点未观察到显著差异。将IMER应用于蛋白质组学工作流程提供了相似的蛋白质鉴定结果、样品分析自动化，并将分析时间减少了15小时。
