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 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.

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Figure 1
Figure 2: Separation of gamma-amino butyric acid A (GABAA) receptors using multi-dimensional gel electrophoresis—blue native (BN) and two-dimensional SDS-PAGE.
Figure 3: Gamma-amino butyric acid A (GABAA)-receptor α-1 subunit identified by multi-enzyme digestion and mass spectrometry.

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Acknowledgements

We acknowledge the contribution by the Verein zur Durchführung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin 'Unser Kind'.

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Correspondence to Gert Lubec.

Supplementary information

Supplementary Figure 1

Spectra of [311 T.AMDWFIAV.C 318], a part of TMD3 in GABAA receptor α-1 subunit, obtained by nano-ESI-LC-MS/MS Qstar XL. (PDF 73 kb)

Supplementary Figure 2

Positive ionization MS/MS spectrum of the peptide m/z 703.58 amu (PDF 16 kb)

Supplementary Table 1

List of identified GABAA receptor α1 subunit peptides (DOC 753 kb)

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Kang, S., Fuchs, K., Sieghart, W. et al. Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains. Nat Protoc 4, 1093–1102 (2009). https://doi.org/10.1038/nprot.2009.92

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