Chong Poh Kuan, Wright Phillip C
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
J Proteome Res. 2005 Sep-Oct;4(5):1789-98. doi: 10.1021/pr0501214.
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.
通过组合分离方法，共鉴定出1399种蛋白质，占嗜热栖热菌P2理论蛋白质组的47%。这包括来自可溶部分的1323种蛋白质、来自不溶部分的44种蛋白质以及来自细胞外或分泌部分的32种蛋白质。对于可溶部分，我们使用传统的二维凝胶电泳（2-DE），对于所有三个细胞部分（可溶、不溶和分泌）则使用鸟枪法蛋白质组学。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，即：（i）利用一维凝胶电泳（1-DE）进行蛋白质分离，随后通过等电聚焦（IEF）进行肽分级分离，以及（ii）蛋白质和肽分级分离均采用IEF。结果表明，进行三次重复质谱分析的一维IEF分级分离工作流程在可溶蛋白质鉴定方面总体结果最佳。使用LC-ESI-MS/MS进行三次进样，蛋白质鉴定数量增加超过50%。还讨论了蛋白质和肽的分级分离效率以及过滤标准。
