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 α1 and His-tagged β3 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% (α1) and 92.9% (β3).