title: Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol.
abstract: 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.
authors: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Huoming
authors: H
authors: Zhang
authors: Qingsong
authors: Q
authors: Lin
authors: Sukumar
authors: S
authors: Ponnusamy
authors: Narasimhan
authors: N
authors: Kothandaraman
authors: Teck Kwang
authors: TK
authors: Lim
authors: Changqing
authors: C
authors: Zhao
authors: Hon Sook
authors: HS
authors: Kit
authors: Biswas
authors: B
authors: Arijit
authors: Mary
authors: M
authors: Rauff
authors: Choy-Leong
authors: CL
authors: Hew
authors: Maxey Ching Ming
authors: MC
authors: Chung
authors: Shashikant B
authors: SB
authors: Joshi
authors: Mahesh
authors: M
authors: Choolani
mesh_terms: D000328:Adult; D002462:Cell Membrane / Q000737:chemistry*; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D000432:Methanol / Q000737:chemistry*; D008969:Molecular Sequence Data; D012997:Solvents / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D012997:Solvents; D014270:Trifluoroethanol; D000432:Methanol