Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins.
A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.
Josic
Djuro
D
Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital, The CORO Center, Providence, RI 02903, USA. Djuro.Josic@brown.edu
Brown
Mari Kino
MK
Huang
Feilei
F
Callanan
Helen
H
Rucević
Marijana
M
Nicoletti
Alison
A
Clifton
James
J
Hixson
Douglas C
DC
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D008099:Liver / Q000166:cytology; D008565:Membrane Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D051381:Rats
D008565:Membrane Proteins