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

通过综合的分离方法，共鉴定出1399种蛋白质，占到了苏铁硫磺菌P2理论蛋白组总量的47%。其中，1323种蛋白质来自可溶性部分，44种来自不溶性部分，32种来自细胞外或分泌部分。对于可溶性部分，我们采用了传统的二维凝胶电泳（2-DE）技术；而对于所有三个细胞部分（可溶性、不溶性及分泌部分），我们使用了射弹蛋白质组学技术。在射弹蛋白质组学中，我们探讨了两种基于凝胶的分级方法：（一）通过一维凝胶电泳（1-DE）进行蛋白质分离，随后采用等电聚焦（IEF）进行肽段分级；（二）同时利用IEF对蛋白质和肽段进行分级。结果表明，采用一维IEF分级流程并结合三次重复质谱分析，在可溶性蛋白质鉴定方面取得了最佳的整体效果。通过三次进样，LC-ESI-MS/MS技术实现了蛋白质鉴定率超过50%的提升。蛋白质和肽段的分级效率，以及过滤标准也在此进行了讨论。
关键词：2-DE • 射弹法 • LC-ESI-MS/MS • 多次进样 • 预分级 • 苏铁硫磺菌