Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains

Sung Ung Kang, Karoline Fuchs, Werner Sieghart, Arnold Pollak, Edina Csaszar, Gert Lubec

Research output: Contribution to journalArticlepeer-review

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 α-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 α-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 ∼10 d to complete and may represent a step forward in the complex analysis of other membrane or hydrophobic proteins.

Original languageEnglish (US)
Pages (from-to)1093-1102
Number of pages10
JournalNature protocols
Volume4
Issue number7
DOIs
StatePublished - Aug 10 2009
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Gel-based mass spectrometric analysis of a strongly hydrophobic GABA<sub>A</sub>-receptor subunit containing four transmembrane domains'. Together they form a unique fingerprint.

Cite this