An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis of Arabidopsis Plasma Membranes
An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis of Arabidopsis Plasma Membranes
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DOI：
10.1021/pr801044y
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发表时间：
2009-06-01
影响因子：
4.4
通讯作者：
Goshe, Michael B.
中科院分区：
文献类型：
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作者：
Mitra, Srijeet K.;Walters, Benjamin T.;Goshe, Michael B.
膜蛋白参与多种细胞过程，是许多信号级联的重要组成部分，但由于其高度疏水性以及在植物中研究这些蛋白质的复杂性，人们正在开发替代方法，以便在蛋白质组范围内更好地表征这些蛋白质。在我们之前的工作中（Mitra, S. K.et al. J. Proteome Res. 2007, 6, (5), 1933-50），与 Brij-58 增溶相比，甲醇辅助增溶被确定有助于识别疏水性和亲水性膜蛋白，并且对于富含亮氨酸的重复受体样激酶 (LRR RLK) 特别有效。为了提高肽的鉴定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，相对于传统固相萃取 (SPE) 使用氯仿萃取，除了有效去除消化中使用的胰蛋白酶之外，还有助于去除叶绿素（植物组织制剂中可能影响下游分析的主要污染物）。根据统计得出的 5% 错误发现率，氯仿提取程序将质膜蛋白独特肽的识别率比 SPE 提高了 70%，这使得膜转运蛋白和 LRR RLK 的检测率增加了近 2 倍，而没有增加对污染性 Rubisco 和核糖体肽的识别率。总的来说，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。
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.