Case Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.
PLoS One. 2010 Dec 30;5(12):e15332. doi: 10.1371/journal.pone.0015332.
Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 µg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic (18)O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.
在这里，我们描述了一种新型的挥发性表面活性剂全氟辛酸 (PFOA) 在鸟枪法蛋白质组学中的应用。结果表明，PFOA 能有效地溶解膜蛋白，其效果与十二烷基硫酸钠 (SDS) 相当。PFOA 浓度高达 0.5%（w/v）时，不会显著抑制胰蛋白酶的活性。PFOA 的独特性质使我们能够开发出一种单管鸟枪法蛋白质组学方法，该方法使用所有挥发性化学物质，在进行质谱分析之前可以很容易地通过蒸发去除。实验步骤包括：1）在 2%PFOA 中提取蛋白质；2）用三乙基膦还原半胱氨酸残基，并使其与碘乙醇发生 S-烷基化；3）在 0.5%PFOA 中用胰蛋白酶消化蛋白质；4）通过蒸发去除 PFOA；5）对得到的肽进行 LC-MS/MS 分析。该方法的通用性通过光感受器外段膜制剂得到了证明。我们从 1µg 酶解肽中鉴定出了 75 种蛋白质，仅在 1 小时的 LC-MS/MS 运行中即可完成。鉴定出的蛋白质中有约 67%被归类为膜蛋白。我们还证明，在去除 PFOA 后可以加入酶切 (18)O 标记程序，用于定量蛋白质组学实验。该方法不需要样品净化设备，如固相萃取和膜过滤器，因此在任何实验步骤中都不会损失蛋白质/肽。因此，这种单管鸟枪法蛋白质组学方法克服了表面活性剂在蛋白质组学实验中的主要缺点。
