Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.
Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.
复制标题
DOI：
10.1074/mcp.m110.001479
复制
发表时间：
2011-02
期刊：
影响因子：
--
通讯作者：
Pasa-Tolić L
中科院分区：
文献类型：
--
作者：
López-Ferrer D;Petritis K;Robinson EW;Hixson KK;Tian Z;Lee JH;Lee SW;Tolić N;Weitz KK;Belov ME;Smith RD;Pasa-Tolić L
整合自上而下自下而上的蛋白质组学与在线消化相结合，具有很大的潜力，以改善生物系统中的蛋白质异构体的表征，并可用于高通量蛋白质组学实验。自下而上的蛋白质组学最终提供了在蛋白质组被消化后从肽的串联MS分析中获得的肽序列。相反，自上而下的蛋白质组学需要完整蛋白质的MS分析，以更有效地表征遗传变异和/或翻译后修饰。在这里，我们描述了最近的努力，有效地整合自下而上和自上而下的LC-MS为基础的蛋白质组学策略。由于大多数蛋白质组学分离利用酸性条件，我们利用胃蛋白酶的相容性（最佳消化条件是在低pH值下）整合到自下而上和自上而下的蛋白质组学工作流程中。成功地进行了压力增强的胃蛋白酶消化，并在使用Barocycler的离线模式或使用被称为快速在线消化系统（FOLDS）的改进的高压LC系统的在线模式下用几种标准蛋白质进行表征。使用胃蛋白酶和整个微生物蛋白质组测试FOLDS，并在同一平台上将结果与传统的胰蛋白酶消化进行比较。此外，FOLDS与RePlay配置集成，以使用蛋白质和猴痘病毒蛋白质组的标准混合物来展示超快速集成的自下而上自上而下的蛋白质组学策略。
Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.