Department of Chemistry, University of Alberta, Edmonton, Canada.
Anal Chim Acta. 2011 Jul 18;698(1-2):36-43. doi: 10.1016/j.aca.2011.04.039. Epub 2011 Apr 28.
Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.
三种表面活性剂辅助的鸟枪法，使用酸不稳定的表面活性剂，3-[3-(1,1-双烷氧基乙基)吡啶-1-基]丙烷-1-磺酸酯(PPS)和 3-[(2-甲基-2-十一烷基-1,3-二恶烷-4-基)-甲氧基]-1-丙磺酸钠(RapiGest)以及十二烷基硫酸钠(SDS)，被研究其在膜蛋白质组分析中的适用性。结果表明，RapiGest 是一种用于处理大肠杆菌和 MCF7 细胞膜蛋白质组的首选试剂，可用于胰蛋白酶消化物的液相色谱串联质谱(LC-MS/MS)分析。与 SDS 和 PPS 方法相比，RapiGest 方法允许鉴定更多的肽和蛋白质，并且没有明显的偏向于由 RapiGest 和 SDS 方法鉴定的肽和蛋白质的类型，而通过 PPS 方法鉴定的亲水性肽和蛋白质的比例略高。SDS 和 PPS 方法在鉴定的肽和蛋白质数量方面性能相似。由于 SDS 方法需要使用强阳离子交换(SCX)等技术去除 SDS，我们进一步研究了 SCX 对通过分析富含大肠杆菌膜部分的消化物以及标准蛋白牛血清白蛋白(BSA)的样品损失的影响。结果表明，在 SCX 清洗步骤中会遇到广泛的样品损失(多达 62%)。然后，我们应用 RapiGest 方法与二维 LC-MS/MS 相结合，对大肠杆菌膜蛋白质组进行了表征。总共鉴定了 1626 种独特的蛋白质(5799 种独特的肽)，肽假阳性率为 2.4%。具有已知细胞位置的鉴定蛋白中约 60%被鉴定为膜蛋白。其中，约 75%为整合膜蛋白。这项工作代表了通过蛋白质组学技术生成的最全面的大肠杆菌膜蛋白质组之一。
