Mitra Srijeet K, Walters Benjamin T, Clouse Steven D, Goshe Michael B
Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609, USA.
J Proteome Res. 2009 Jun;8(6):2752-67. doi: 10.1021/pr801044y.
Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.
膜蛋白参与多种细胞过程，是许多信号级联反应的重要组成部分。然而，由于其高度疏水的性质以及在植物中研究这些蛋白的复杂性，人们正在开发替代方法，以便在全蛋白质组范围内更好地表征这些蛋白。在我们之前的工作中（米特拉，S.K.等人，《蛋白质组研究杂志》，2007年，6卷，第5期，1933 - 50页），与Brij - 58增溶法相比，甲醇辅助增溶被确定有助于鉴定疏水和亲水膜蛋白，并且对富含亮氨酸重复序列的类受体激酶（LRR RLK）特别有效。为了提高肽段鉴定率并克服胰蛋白酶消化后的样品损失，我们开发了一种有效的氯仿提取方法来促进质膜蛋白的鉴定。在离线强阳离子交换液相色谱（SCXC）和反相液相色谱 - 串联质谱（LC/MS/MS）分析之前，使用氯仿提取代替传统的固相萃取（SPE），除了有效去除消化中使用的胰蛋白酶外，还促进了叶绿素的去除，叶绿素是植物组织制备中的主要污染物，会影响下游分析。基于统计学得出的5%错误发现率，氯仿提取程序使质膜蛋白独特肽段的鉴定比SPE增加了70%，这使得膜转运蛋白和LRR RLK的检测增加了近2倍，同时不会增加污染性的核酮糖 - 1,5 - 二磷酸羧化酶（Rubisco）和核糖体肽段的鉴定。总体而言，甲醇和氯仿的联合使用提供了一种研究膜蛋白的有效方法，并且可以很容易地应用于其他组织和细胞类型进行蛋白质组分析。
