van Oostveen I, Ducret A, Aebersold R
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA.
Anal Biochem. 1997 May 1;247(2):310-8. doi: 10.1006/abio.1997.2052.
Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.
用于通过氨基酸测序或通过将质谱数据与序列数据库相关联来鉴定蛋白质的质谱技术正变得越来越灵敏，并且正在迅速接近通过对凝胶中的蛋白质进行染色或在电印迹后对膜上的蛋白质进行染色所达到的检测极限。在此，我们介绍一种对电印迹到硝酸纤维素或聚偏二氟乙烯膜上的蛋白质进行灵敏染色的技术，以及用于此类蛋白质的酶切条件，以实现肽的最佳回收率。该技术基于在膜结合蛋白上沉积胶体银。在膜上通过蛋白水解产生的肽混合物以高产率回收，并且与反相色谱分析和在线电喷雾电离质谱兼容。这种简单快速的胶体银染色程序能够使条带中少于5 ng的蛋白质可视化，从而接近凝胶中银染色的灵敏度。我们证明，该方法能够检测亚皮摩尔量的电印迹蛋白质，并通过高效液相色谱 - 电喷雾电离串联质谱对其进行鉴定。
