Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol.
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.
Zhang
Huoming
H
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Lin
Qingsong
Q
Ponnusamy
Sukumar
S
Kothandaraman
Narasimhan
N
Lim
Teck Kwang
TK
Zhao
Changqing
C
Kit
Hon Sook
HS
Arijit
Biswas
B
Rauff
Mary
M
Hew
Choy-Leong
CL
Chung
Maxey Ching Ming
MC
Joshi
Shashikant B
SB
Choolani
Mahesh
M
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*
D008565:Membrane Proteins; D012997:Solvents; D014270:Trifluoroethanol; D000432:Methanol