Lin Yong, Wang Kunbo, Liu Zhonghua, Lin Haiyan, Yu Lijun
National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, PR China; Hunan Collaborative Innovation Center for Utilization of Functional Ingredients from Botanicals, Hunan Agricultural University, Changsha 410128, PR China.
National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, PR China; Key Laboratory of Tea Science of Ministry of Education, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Oct 1;1002:144-51. doi: 10.1016/j.jchromb.2015.08.019. Epub 2015 Aug 21.
Despite the biological importance of membrane proteins, their analysis has lagged behind that of soluble proteins and still presents a great challenge mainly because of their highly hydrophobic nature and low abundance. Sodium deoxycholate (SDC)-assisted digestion strategy has been introduced in our previous papers, which cleverly circumvents many of the challenges in shotgun membrane proteomics. However, it is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of 1% SDC. In this study, an enhanced SDC-assisted digestion method (ESDC method) was developed that incorporates the almost strongest ability of SDC with a high concentration (5%) to lyse membrane and extract/solubilize hydrophobic membrane proteins, and then dilution to 1% for more efficient digestion. The comparative study using rat liver membrane-enriched sample showed that, compared with previous SDC-assisted method and the "universal" filter-aided sample preparation (FASP) method, the ESDC method not only increased the identified number of total proteins, membrane proteins, hydrophobic proteins, integral membrane proteins (IMPs) and IMPs with more than 5 transmembrane domains (TMDs) by an average of 10.8%, 13.2%, 17.8%, 17.9% and 52.9%, respectively, but also enhanced the identified number of total peptides and hydrophobic peptides by averagely 12.5% and 14.2%. These results demonstrated that the ESDC method provides a substantial improvement in the recovery and identification of membrane proteins, especially those with high hydrophobicity and multiple TMDs, and thereby displaying more potential for shotgun membrane proteomics.
尽管膜蛋白具有重要的生物学意义，但其分析仍落后于可溶性蛋白，并且仍然面临巨大挑战，主要原因在于其高度疏水的性质和低丰度。在我们之前的论文中引入了脱氧胆酸钠（SDC）辅助消化策略，该策略巧妙地规避了鸟枪法膜蛋白质组学中的许多挑战。然而，由于1% SDC的提取/溶解能力稍弱，它会导致显著的样品损失。在本研究中，开发了一种增强的SDC辅助消化方法（ESDC方法），该方法将高浓度（5%）的SDC裂解膜和提取/溶解疏水膜蛋白的几乎最强能力与随后稀释至1%以实现更高效消化相结合。使用大鼠肝脏富含膜的样品进行的比较研究表明，与先前的SDC辅助方法和“通用”的滤膜辅助样品制备（FASP）方法相比，ESDC方法不仅使总蛋白、膜蛋白、疏水蛋白、整合膜蛋白（IMP）和具有超过5个跨膜结构域（TMD）的IMP的鉴定数量分别平均增加了10.8%、13.2%、17.8%、17.9%和52.9%，而且还使总肽段和疏水肽段的鉴定数量分别平均增加了12.5%和14.2%。这些结果表明，ESDC方法在膜蛋白的回收率和鉴定方面有显著改进，尤其是对于具有高疏水性和多个TMD的膜蛋白，从而在鸟枪法膜蛋白质组学中显示出更大的潜力。
