Yan J X, Wait R, Berkelman T, Harry R A, Westbrook J A, Wheeler C H, Dunn M J
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Center, Harefield Hospital, Middlesex, UK.
Electrophoresis. 2000 Nov;21(17):3666-72. doi: 10.1002/1522-2683(200011)21:17<3666::AID-ELPS3666>3.0.CO;2-6.
The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.
随着基因组序列信息的日益丰富，以及分析方法的改进，实现了高通量、高灵敏度的蛋白质鉴定。银染仍然是用于二维凝胶电泳（2-D PAGE）分离蛋白质可视化的最灵敏方法。已经开发了几种银染方案，这些方案与后续的质谱分析具有更好的兼容性。我们描述了一种改良的银染方法，该方法作为商业试剂盒（Silver Stain PlusOne；安玛西亚 Pharmacia 生物技术公司，英国阿默舍姆）提供。用这种改良方案获得的二维图谱与其他银染方法的图谱相当。省略敏化试剂可实现更高上样量而不会饱和，这有利于蛋白质鉴定和定量。我们表明，通过改良染色可视化的蛋白质经胰蛋白酶消化后，通过基质辅助激光解吸/电离和串联电喷雾电离均可获得出色的质谱图。我们得出结论，改良的银染方案与后续的质谱分析具有高度兼容性。
