Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins.
Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.
Deshusses
Jacques M P
JM
Biomedical Proteomics Group, Central Clinical Chemistry Laboratory, Geneva University Hospital, Rue Micheli-du-Crest 24, CH-1211 Geneva, Switzerland. jacques.deshusses@dim.hcuge.ch
Burgess
Jennifer A
JA
Scherl
Alexander
A
Wenger
Yvan
Y
Walter
Nadia
N
Converset
Véronique
V
Paesano
Salvo
S
Corthals
Garry L
GL
Hochstrasser
Denis F
DF
Sanchez
Jean-Charles
JC
D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014270:Trifluoroethanol / Q000737:chemistry*
D008565:Membrane Proteins; D014270:Trifluoroethanol