Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry.
Comparative analysis has long been utilized in biological research to interpret protein interactions in both drug naïve versus drug challenged and normal versus diseased tissues. The technology of proteomics today allows researchers to provide insight into old and still open questions related to biological mechanisms while offering the opportunity to discover novel details in cellular lifecycles. Perhaps the most powerful way to execute these differential displays is in the combination of two-dimensional (2-D) gel electrophoresis and mass spectrometry. While these two techniques together are well suited for abundant and soluble proteins found in cells, rare proteins and integral membrane proteins are still problematic. Recently, a series of novel zwitterionic detergents has been reported in the literature that shows a substantial improvement in solubilizing integral membrane proteins. We show that the amidosulfobetaine, 4-octylbenzol amidosulfobetaine, is better than 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) at solubilizing both an ion channel and a G-protein coupled receptor (GPCR), while another amidosulfobetaine, myristic amidosulfobetaine (ASB-14), was better than CHAPS at solubilizing a GPCR. Neither membrane protein was visible after staining with colloidal Coomassie blue, silver nor Sypro Ruby. However, a comparison against a duplicate immunoblot allowed for the localization and identification of the ion channel from a 2-D gel by liquid chromatography-tandem mass spectrometry.
Henningsen
Robert
R
Neurobiology Unit, Roche Bioscience, Palo Alto, CA 94304, USA.
Gale
Brenda L
BL
Straub
Kenneth M
KM
DeNagel
Diane C
DC
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003902:Detergents / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007473:Ion Channels / Q000032:analysis / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D018048:Receptors, Purinergic P2 / Q000032:analysis / Q000737:chemistry; D058477:Receptors, Purinergic P2X3; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003902:Detergents; D007473:Ion Channels; D008565:Membrane Proteins; D010446:Peptide Fragments; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3