Gel-Based Mass Spectrometric Analysis of Recombinant GABA<sub>A</sub> Receptor Subunits Representing Strongly Hydrophobic Transmembrane Proteins
Sung Ung Kang
Karoline Fuchs
Werner Sieghart
Gert Lubec
10.1021/pr800236u.s001
https://acs.figshare.com/articles/journal_contribution/Gel_Based_Mass_Spectrometric_Analysis_of_Recombinant_GABA_sub_A_sub_Receptor_Subunits_Representing_Strongly_Hydrophobic_Transmembrane_Proteins/2924500
GABA<sub>A</sub> 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<sub>A</sub> 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<sub>A</sub> receptor subunits were well-separated and unambiguously identified by sequence coverage of 99.1% (α1) and 92.9% (β3).
2008-08-01 00:00:00
transmitter receptors
GABAA receptors
affinity chromatography
subunit classes
insect cells
sequence coverage
protein subunits
brain tissue
Hydrophobic Transmembrane ProteinsGABAA receptors
multienzyme proteolysis
Recombinant GABAA Receptor Subunits
GABAA receptor subunits