Identification and Characterization of the Sulfolobus solfataricus P2 Proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC?ESI?MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed. Keywords: 2-DE ? shotgun ? LC?MS/MS ? multiple injections ? pre-fractionation ? S. solfataricus

通过联合分离策略，共鉴定出1399种蛋白质，占硫化叶菌Sulfolobus solfataricus P2理论蛋白质组的47%。其中可溶组分包含1323种蛋白质，不溶组分44种，胞外或分泌组分32种。我们针对可溶组分采用常规二维凝胶电泳（2-dimensional gel electrophoresis, 2-DE），并对全部三类细胞组分（可溶、不溶及分泌组分）采用鸟枪法蛋白质组学（shotgun proteomics）。针对鸟枪法蛋白质组学，探索了两种基于凝胶的分级分离方法：(i) 先通过一维凝胶电泳（1-dimensional gel electrophoresis, 1-DE）分离蛋白质，再通过等电聚焦（iso-electric focusing, IEF）分级分离肽段；(ii) 蛋白质与肽段的分级分离均采用等电聚焦。研究结果表明，搭配三次重复质谱分析的1D-IEF分级分离流程，在可溶蛋白质鉴定方面取得了最优的整体效果。采用液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）进行三次进样后，蛋白质鉴定数量实现了50%以上的提升。此外还讨论了蛋白质与肽段的分级分离效率以及过滤筛选标准。关键词：二维凝胶电泳（2-DE）、鸟枪法蛋白质组学、液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）、多次进样、预分级分离、硫化叶菌（S. solfataricus）