Nebrich Grit, Herrmann Marion, Sagi Dijana, Klose Joachim, Giavalisco Patrick
Institute for Human Genetics Humboldt-University Berlin, Charité, Berlin, Germany.
Electrophoresis. 2007 May;28(10):1607-14. doi: 10.1002/elps.200600656.
The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.
对于蛋白质组研究的成功而言，具备易于操作、灵敏且经济高效的二维电泳蛋白质染色方案，并与后续质谱分析具有高度兼容性，仍然是一个先决条件。在本文中，我们描述了一种基于灵敏、均一且与质谱兼容的硝酸银蛋白质染色的快速且易于使用的方法方案，结合胶内消化，采用密理博96孔ZipPlate系统进行肽段制备。通过将192个考马斯亮蓝染色的蛋白质点与其在硝酸银染色的二维电泳凝胶上的对应点进行分析，在真实组织样本上评估了与其他情况下与质谱兼容性较弱的硝酸银染色相比，所生成的蛋白质消化物在质量和质谱兼容性方面的改善情况。此外，通过大规模基质辅助激光解吸电离飞行时间质谱实验的分析评估并证明了该实验设置的适用性，在该实验中，我们分析了来自小鼠肝脏和小鼠脑组织二维电泳凝胶上另外约1000个蛋白质点。
