title: Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains.
abstract: The analysis of highly hydrophobic proteins is still an analytical challenge. Using a recombinant gamma-aminobutyric acid A (GABAA)-receptor subunit as a model protein, we developed a gel-based proteomic approach for high MS/MS-peptide sequence coverage identification. Protein samples were separated by multi-dimensional gel electrophoresis and the three protein spots representing the GABAA-receptor subunit alpha-1 from the last electrophoretic step were used for in-gel digestion with trypsin, chymotrypsin and subtilisin, followed by subsequent mass-spectrometric identification by nano-ESI-LC-MS/MS Qstar XL (quadrupole time-of-flight (qQTOF)) and linear ion trap (LIT) LTQ XL identification. This protocol allows the unambiguous identification of the GABAA-receptor alpha-1 subunit protein with 100% sequence coverage, thus covering all four hydrophobic transmembrane domains. This protocol differs from other methods in the selection of enzymes, digestion conditions and use of the two mass spectrometry principles. The protocol takes approximately 10 d to complete and may represent a step forward in the complex analysis of other membrane or hydrophobic proteins.
authors.affiliation: Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
authors.forename: Sung-Ung
authors.initials: SU
authors.lastname: Kang
authors.forename: Karoline
authors.initials: K
authors.lastname: Fuchs
authors.forename: Werner
authors.initials: W
authors.lastname: Sieghart
authors.forename: Arnold
authors.initials: A
authors.lastname: Pollak
authors.forename: Edina
authors.initials: E
authors.lastname: Csaszar
authors.forename: Gert
authors.initials: G
authors.lastname: Lubec
mesh_terms: D000818:Animals; D005782:Gels / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry*
chemical_list: D005782:Gels; D008565:Membrane Proteins; D011963:Receptors, GABA-A
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