Identification of Trichloroethanol Visualized Proteins from Two-Dimensional Polyacrylamide Gels by Mass Spectrometry
作者:Carol L. Ladner、Robert A. Edwards、David C. Schriemer、Raymond J. Turner
DOI:10.1021/ac051851y
日期:2006.4.1
Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC−MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC−MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.
通过基质辅助激光解吸/电离飞行时间 (MALDI-TOF) 质谱 (MS) 和 MS/MS 有效鉴定二维电泳凝胶上 2,2,2-三氯乙醇 (TCE) 可视化的蛋白质。在之前的研究中,开发了一种将 TCE 置于聚丙烯酰胺凝胶中的方法,以便在 5 分钟内无需染色即可可视化蛋白质条带。 TCE 与色氨酸反应生成可见荧光团,从而实现蛋白质可视化。在本研究中,MALDI-TOF MS 和 LC−MS/MS 用于鉴定随机选择的大肠杆菌蛋白。将 TCE 可视化蛋白质的鉴定与大肠杆菌蛋白质二维凝胶电泳中考马斯亮蓝 (CBB) 染色蛋白质的鉴定进行比较。这项研究表明,TCE 可视化蛋白质与 MALDI-TOF 肽质量指纹法的蛋白质鉴定兼容。对于 10 个随机选择的点,TCE 可视化导致 5 种蛋白质的统计显着性鉴定,CBB 可视化导致 6 种蛋白质的鉴定。 TCE 可视化蛋白质也被证明非常适合使用 LC−MS/MS 进行蛋白质鉴定。在选择进行 MS/MS 分析的 16 个点中,TCE 样品鉴定出 79 种肽;而 CBB 样品则鉴定出 65 种肽。 TCE 样品还支持鉴定出更多蛋白质。色氨酸残基标记的低化学计量不需要在数据库搜索中包含这种修饰。除了与 MS 兼容的快速可视化技术之外,TCE 可视化还利用快速清洗条件对从聚丙烯酰胺凝胶上切下的蛋白质斑点进行样品制备。