Zhang Huoming, Lin Qingsong, Ponnusamy Sukumar, Kothandaraman Narasimhan, Lim Teck Kwang, Zhao Changqing, Kit Hon Sook, Arijit Biswas, Rauff Mary, Hew Choy-Leong, Chung Maxey Ching Ming, Joshi Shashikant B, Choolani Mahesh
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Proteomics. 2007 May;7(10):1654-63. doi: 10.1002/pmic.200600579.
Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.
细胞膜蛋白质组分析受到膜固有疏水性的限制。传统的膜蛋白提取技术使用去污剂、离液剂和有机酸，这些方法需要对样品进行清理或调节pH值，并且会导致大量样品损失。我们使用甲醇（MeOH）、三氟乙醇（TFE）和尿素从红细胞（RBC）中提取膜蛋白，并使用二维液相色谱结合基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF-MS）鉴定膜蛋白。我们发现，基于甲醇和三氟乙醇的有机溶剂方法在膜蛋白分析效率上与尿素相当，并且在亲水性和疏水性肽段的回收方面具有互补性。基于三氟乙醇的方法鉴定出的肽段的平均亲水性总平均值（GRAVY）为-0.107，显著高于基于甲醇的方法（-0.465）（p<0.001）。甲醇和三氟乙醇这两种有机溶剂的顺序使用和辅助使用增加了鉴定出的蛋白质数量及其鉴定的可信度。我们证明了这种策略对于鸟枪法膜蛋白质组分析是有效的。
