The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, PR China.
Electrophoresis. 2009 Dec;30(23):4109-17. doi: 10.1002/elps.200900391.
Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel-embedment and improved in-gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45 degrees C), and the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25 degrees C, gel-embedment at 45 degrees C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome.
分析整体膜蛋白（IMPs）因其疏水性和低丰度而带来特殊挑战。本研究开发了一种新方法，涉及热凝胶嵌入和改进的胶内消化。含有去污剂的膜蛋白裂解液与丙烯酰胺溶液混合，当凝胶聚合时，蛋白质被嵌入。为了进行比较，在不同温度（25、35 或 45°C）下进行蛋白质嵌入，并分别在 0.1% RapiGest 试剂（ALS）、0.1%脱氧胆酸钠和 10%ACN 的存在下进行胶内消化。所得肽段通过毛细管液相色谱-串联质谱进行提取和分析。与 25°C 相比，45°C 的凝胶嵌入提高了蛋白质嵌入效率，从而增加了 27%的蛋白质鉴定。0.1%脱氧胆酸钠在提高胶内嵌入蛋白的消化和胰蛋白酶消化回收方面比 0.1%ALS 和 10%ACN 更有效，特别是对疏水性 IMPs。通过热凝胶嵌入结合改进的胶内消化策略鉴定的 326 个 IMPs 中，有 149 个（46%）蛋白至少有两个映射的跨膜结构域。这些结果表明，我们新开发的方案可以促进整体膜蛋白质组的高通量分析。
