Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis T, Mann M
Protein & Peptide Group, EMBL, Heidelberg, Germany.
Nature. 1996 Feb 1;379(6564):466-9. doi: 10.1038/379466a0.
Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
对复杂生物结构和过程进行分子分析越来越需要用于蛋白质测序的灵敏方法。电喷雾质谱已应用于短肽的高灵敏度测序，但技术难题阻碍了在凝胶分离蛋白方面取得类似的成功。本文我们报道了一种简单且可靠的技术，利用纳米电喷雾串联质谱对通过聚丙烯酰胺凝胶电泳分离的蛋白质进行测序。考马斯亮蓝或银染凝胶上起始蛋白质材料低至5 ng即可进行测序。可获得长达16个氨基酸的多个序列片段，若该蛋白质已存在于数据库中，则可明确鉴定该蛋白质，或提供信息用于克隆相应基因。我们已将此方法应用于一种蛋白质的测序和克隆，该蛋白质在体外抑制毛细血管内皮细胞的增殖，因此可能对实体瘤具有潜在的抗血管生成作用。
