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.
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The use of Lobind tubes significantly reduces the binding of hydrophobic proteins to the wall of the tubes.
“as the use of these tubes significantly reduces the binding of hydrophobic proteins to the wall of the tubes (37).”
From Enhanced mTORC1 signaling and Protein Synthesis in Parkinson’s Disease Pathogenesis Disease Pathogenesis (2022) · cites this paper as [37]
Cites
Baer, A. S., Syed, Y. A., Kang, S. U., Mitteregger, D., Vig, R., ffrench-Constant, C., Franklin, R. J. M., Altmann, F., Lubec, G., & Kotter, M. R. (2009) · Brain · doi:10.1093/brain/awn334 · Cited by 211 · see citing works
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Khan, M. R., Yin, X., Kang, S.-U., Mitra, J., Wang, H., Ryu, T., Brahmachari, S., Karuppagounder, S. S., Kimura, Y., Jhaldiyal, A., et al. (2023) · Science Translational Medicine · doi:10.1126/scitranslmed.add0499 · Cited by 64 · see citing works
Self-citationGhafari, M., Falsafi, S. K., Szodorai, E., Kim, E.-J., Li, L., Höger, H., Berger, J., Fuchs, K., Sieghart, W., & Lubec, G. (2017) · Brain Structure and Function · doi:10.1007/s00429-016-1233-x · Cited by 19 · see citing works
Self-citationKhan, M. R., Yin, X., Kang, S.-U., Mitra, J., Wang, H., Brahmachari, S., Karuppagounder, S. S., Kimura, Y., Jhaldiyal, A., Kim, H. H., et al. (2022) · doi:10.1101/2022.10.03.510455
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- Authors
- Kang, S.-U., Fuchs, K., Sieghart, W., Pollak, A., Csaszar, E., & Lubec, G.
- Affiliations
- University of Vienna (AT)
- Medical University of Vienna (AT)
- Max Perutz Labs (AT)
- Venue
- Nature Protocols
- Published
- 2009-07
- Volume
- 4
- Issue
- 7
- Pages
- 1093-1102
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2064940913
- Cited by
- 51
- References
- 1
- Citations by year
- 2023: 1 · 2022: 2 · 2021: 1 · 2016: 5 · 2015: 4 · 2014: 6 · 2013: 5 · 2012: 8
- FWCI
- 3.86
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- hydrophobic proteins, membrane proteins, transmembrane domain, in-gel digestion, trypsin, chymotrypsin, subtilisin, peptide sequence coverage, protein identification
- MeSH terms
- Animals, Gels, Membrane Proteins, Receptors, GABA-A, Mass Spectrometry, Protein Structure, Tertiary, Rats, Hydrophobic and Hydrophilic Interactions
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