Figeys D, Gygi S P, Zhang Y, Watts J, Gu M, Aebersold R
Department of Molecular Biotechnology, University of Washington, Seattle, USA.
Electrophoresis. 1998 Jul;19(10):1811-8. doi: 10.1002/elps.1150191045.
Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.
分析性和制备性电泳分离技术一直是蛋白质生物化学以及整个生物科学领域的重要工具。高分辨率电泳技术与高性能分析程序的结合极大地推动了分析性蛋白质生物化学的发展。在本报告中，我们描述了电泳分离技术与电喷雾电离（ESI）串联质谱（MS/MS）的结合。在鉴定从高皮摩尔到低飞摩尔量蛋白质的背景下，介绍了一系列不同的技术，包括自动高效液相色谱（HPLC）-MS/MS、毛细管-HPLC-MS/MS以及固相萃取（SPE）-毛细管区带电泳（CZE）-MS/MS。展示了这些强大的新工具在大规模蛋白质组分析中的应用。此外，还介绍了正交分离技术的组合，如固定化金属亲和色谱（IMAC）与SPE-CZE-MS/MS的组合，以及先进行IMAC然后进行HPLC，再进行SPE-CZE-MS/MS的组合，用于详细研究特定蛋白质的翻译后修饰。
