Liberal Arts Education Center, Tokai University, Minamiaso, Kumamoto, Japan.
Proteomics. 2013 Feb;13(3-4):457-66. doi: 10.1002/pmic.201200272. Epub 2013 Jan 14.
Proteolysis is a key step in proteomic studies integrated with MS analysis but the conventional method of in-solution digestion is limited by time-consuming procedures and low sensitivity. Furthermore, obtaining reliable peptide maps and meaningful sequence data using MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions. Recently, various immobilized-enzyme reactors have been developed for highly efficient proteolysis in MS-based proteomic analysis. This review focuses on the proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. We describe the preparation of enzyme reactors by several techniques and protein digestion under unusual conditions. Analysis of posttranslational modifications by enzyme reactors prepared using our immobilization method is presented as a model application. Analysis systems using immobilized-enzyme reactors are expected to become useful tools for proteomic studies and diverse applications in biotechnology.
蛋白水解是与 MS 分析相结合的蛋白质组学研究中的关键步骤，但传统的溶液内消化方法受到耗时的程序和低灵敏度的限制。此外，使用 MS 分析获得可靠的肽图和有意义的序列数据不仅需要消化肽的分离，还需要严格定义的蛋白水解条件。最近，已经开发了各种固定化酶反应器，用于 MS 基蛋白质组分析中的高效蛋白水解。本综述重点介绍使用蛋白酶固定化反应器进行蛋白水解和快速分析蛋白质序列。我们描述了通过几种技术制备酶反应器以及在不寻常条件下进行蛋白水解的方法。通过使用我们的固定化方法制备的酶反应器进行翻译后修饰分析作为模型应用进行介绍。使用固定化酶反应器的分析系统有望成为蛋白质组学研究和生物技术中各种应用的有用工具。
