Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels
Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels
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DOI：
10.1021/ac950914h
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发表时间：
1996-03-01
影响因子：
7.4
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Shevchenko, A;Wilm, M;Mann, M
来自银染凝胶的蛋白质可以被酶消化，并且通过质谱法分析和测序所产生的肽。当从考马斯和银染凝胶中提取时，标准蛋白质产生相同的肽图谱，如通过电喷雾和MALDI质谱法所判断的。通过本文所述的方案可以达到低纳克范围，并且该方法具有耐用性。银染色的一维凝胶从酵母蛋白质的馏分进行了分析，通过纳米电喷雾串联质谱。在测序中，覆盖了超过1000个氨基酸，由于银染色程序，没有化学修饰的证据，银染色可以大大缩短样品制备时间，因此可能优于考马斯染色。这项工作消除了聚丙烯酰胺凝胶上分离的蛋白质的低水平序列分析的主要障碍。
Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry, The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure, Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.