Winkler Christiane, Denker Katrin, Wortelkamp Stefanie, Sickmann Albert
Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
Electrophoresis. 2007 Jun;28(12):2095-9. doi: 10.1002/elps.200600670.
Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.
聚丙烯酰胺凝胶电泳（PAGE）的染色方案必须灵敏，且不应影响对所选样品的进一步质谱分析。在本研究中，系统地阐明了不同银染和考马斯亮蓝染色方案与纳升液相色谱-串联质谱（nano-LC-MS/MS）系统的质谱兼容性。总共使用了13种不同的银染、1种咪唑染色和2种考马斯亮蓝染色方案，并就其实现的序列覆盖率和检测灵敏度进行了相互比较。使用三种蛋白质作为模型蛋白（牛血清白蛋白、兔L-乳酸脱氢酶、牛乳β-乳球蛋白），浓度依次降低（从12皮摩尔降至30飞摩尔），并应用不同的染色方案。本研究的结论是，两种银染方案（Blum, H.等人，《电泳》，1987年，第8卷，第93 - 99页；Shevchenko, A.等人，《分析化学》，1996年，第68卷，第850 - 858页）兼具良好的序列覆盖率和灵敏度，推荐用于nano-LC-MS/MS分析。
