Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Dec 1;878(31):3259-66. doi: 10.1016/j.jchromb.2010.10.005. Epub 2010 Oct 16.
In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.
在蛋白质组学研究领域，膜蛋白的分析仍然面临许多技术挑战。在这个难题中，一个基本问题是如何保持适当的溶剂环境，以使疏水性蛋白质保持溶解状态。我们提出，在高浓度尿素溶液中使膜蛋白变性，使它们能够离子化，从而可以采用离子交换层析来分离它们。从鼠肝中制备的膜蛋白溶解在 6M 盐酸胍、20mM Tris-HCl、pH9.0 中，并加载到串联层析装置上，该装置与 Q-Sepharose FF 和 Sephacryl S-200HR 偶联。这些柱子能够吸附 97.87%的膜蛋白制剂。使用线性 NaCl（0-1.0M）梯度，在 0.1-1.0M NaCl 洗脱结合的蛋白质，并通过 SDS-PAGE 进行检查。此外，蛋白质条带经胰蛋白酶切割和消化后，进行反相色谱分离消化肽和离子阱质谱鉴定蛋白质。从 SDS-PAGE 凝胶中，相邻条带之间的蛋白质重叠仅为 21.34%，表明阴离子排阻偶联层析有效地分离了这些膜蛋白。在鉴定的 392 种蛋白质中，有 306 种是膜蛋白或膜相关蛋白。根据疏水性的计算，83 种蛋白质的 GRAVY 评分大于或等于 0.00。综合所有这些证据，我们的结果表明，这种方法对于研究鼠肝的膜蛋白质组是令人满意的。
