Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits representing strongly hydrophobic transmembrane proteins.
GABA(A) receptors are the major inhibitory transmitter receptors in mammalian brain and are composed of several protein subunits that can belong to different subunit classes, leading to enormous heterogeneity. To establish techniques for the analysis of GABA(A) receptors in complex mixtures such as brain tissue, recombinant receptors composed of alpha1 and His-tagged beta3 subunits expressed in insect cells were purified by affinity chromatography and run on blue native gels. After denaturing, receptors were subjected to one- or two-dimensional electrophoresis in SDS-gels. Proteins were cleaved by multienzyme proteolysis and subjected to nano-ESI-LC-MS/MS. Both GABA(A) receptor subunits were well-separated and unambiguously identified by sequence coverage of 99.1% (alpha1) and 92.9% (beta3).
Kang
Sung Ung
SU
Department of Pediatrics, Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Austria.
Fuchs
Karoline
K
Sieghart
Werner
W
Lubec
Gert
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D005782:Gels; D057927:Hydrophobic and Hydrophilic Interactions; D007313:Insecta / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis*; D021122:Protein Subunits / Q000032:analysis; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D005782:Gels; D008565:Membrane Proteins; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins