Castellanos-Serra L, Proenza W, Huerta V, Moritz R L, Simpson R J
Center for Genetic Engineering and Biotechnology (CIGB) Havana, Cuba.
Electrophoresis. 1999 Apr-May;20(4-5):732-7. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<732::AID-ELPS732>3.0.CO;2-Q.
Identification and characterization of proteins isolated from natural sources by polyacrylamide gel electrophoresis has become a routine technique. However, efficient sample proteolysis and subsequent peptide extraction is still problematic. Here, we present an improved protocol for the rapid detection of polyacrylamide gel-separated proteins, in situ protein modification, proteolytic digestion and peptide extraction for subsequent protein identification and characterization by capillary high-performance liquid chromatography/tandem mass spectrometry. This simple technique employs the rapid imidazole-zinc reverse stain, in-gel S-pyridylethylation and proteolytic digestion of microcrushed polyacrylamide gel pieces with proteases. This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation.
通过聚丙烯酰胺凝胶电泳从天然来源分离蛋白质并进行表征已成为一项常规技术。然而，高效的样品蛋白水解及随后的肽段提取仍然存在问题。在此，我们提出了一种改进方案，用于快速检测聚丙烯酰胺凝胶分离的蛋白质、原位蛋白质修饰、蛋白水解消化以及肽段提取，以便随后通过毛细管高效液相色谱/串联质谱对蛋白质进行鉴定和表征。这项简单技术采用快速咪唑 - 锌反向染色、凝胶内S - 吡啶基乙基化以及用蛋白酶对微粉碎的聚丙烯酰胺凝胶片进行蛋白水解消化。由于所有样品处理步骤均在接近中性的pH条件下进行，该技术无需进行缓冲液交换或凝胶冻干，因此很容易实现自动化。
