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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Kashino, Y. Separation methods in the analysis of protein membrane complexes. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 797, 191–216 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Speers, A.E. & Wu, C.C. Proteomics of integral membrane proteins–theory and application. Chem. Rev. 107, 3687–3714 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Rabilloud, T., Chevallet, M., Luche, S. & Lelong, C. Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update. Proteomics 8, 3965–3973 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Zahedi, R.P., Moebius, J. & Sickmann, A. Two-dimensional BAC/SDS-PAGE for membrane proteomics. Subcell. Biochem. 43, 13–20 (2007).

    Article  Google Scholar 

  5. 5

    Braun, R.J., Kinkl, N., Beer, M. & Ueffing, M. Two-dimensional electrophoresis of membrane proteins. Anal. Bioanal. Chem. 389, 1033–1045 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Barnard, E.A. et al. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291–313 (1998).

    CAS  Google Scholar 

  7. 7

    Sieghart, W. & Sperk, G. Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr. Top. Med. Chem. 2, 795–816 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Sarter, M., Schneider, H.H. & Stephens, D.N. Treatment strategies for senile dementia: antagonist beta-carbolines. Trends Neurosci. 11, 13–17 (1988).

    CAS  Article  Google Scholar 

  9. 9

    Paulsen, O. & Moser, E.I. A model of hippocampal memory encoding and retrieval: GABAergic control of synaptic plasticity. Trends Neurosci. 21, 273–278 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Macdonald, R.L. & Olsen, R.W. GABAA receptor channels. Annu. Rev. Neurosci. 17, 569–602 (1994).

    CAS  Article  Google Scholar 

  11. 11

    Sieghart, W. & Ernst, M. Heterogeneity of GABA-A receptors: revived interest in the development of subtype-selective drugs. Curr. Med. Chem-Centr. Nervous Syst. 5, 217–242 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Akentieva, N.P. et al. Society for Neurosciences, Meeting Atlanta, Abstract 527.521 (2006).

  13. 13

    Schindler, J., Lewandrowski, U., Sickmann, A., Friauf, E. & Nothwang, H.G. Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning. Mol. Cell Proteomics 5, 390–400 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Kang, S.U., Fuchs, K., Sieghart, W. & Lubec, G. Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits representing strongly hydrophobic transmembrane proteins. J. Proteome Res. 7, 3498–3506 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Baer, A.S. et al. Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. Brain 132, 465–481 (2009).

    Article  Google Scholar 

  16. 16

    Chen, W.Q., Kang, S.U. & Lubec, G. Protein profiling by the combination of two independent mass spectrometry techniques. Nat. Protoc. 1, 1446–1452 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Bierczynska-Krzysik, A., Kang, S.U., Silberrring, J. & Lubec, G. Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins. Neurochem. Int. 49, 245–255 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Oberacher, H. et al. On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library: 2. Optimization and characterization of the search algorithm. J. Mass Spectrom. 44, 485–493 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Barrera, N.P. et al. Atomic force microscopy reveals the stoichiometry and subunit arrangement of the alpha4beta3delta GABA(A) receptor. Mol. Pharmacol. 73, 960–967 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Knight, A.R., Stephenson, F.A., Tallman, J.F. & Ramabahdran, T.V. Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors. Receptors Channels 7, 213–226 (2000).

    CAS  PubMed  Google Scholar 

  21. 21

    Louiset, E., McKernan, R., Sieghart, W. & Vaudry, H. Subunit composition and pharmacological characterization of gamma-aminobutyric acid type A receptors in frog pituitary melanotrophs. Endocrinology 141, 1083–1092 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Rapallino, M.V. et al. Immunocytochemical study of alpha 1 and beta 2/3 subunits of GABAA receptors in freehand isolated vestibular Deiters' neurons. Receptors Channels 9, 77–81 (2003).

    CAS  PubMed  Google Scholar 

  23. 23

    Lubec, G. & Afjehi-Sadat, L. Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev. 107, 3568–3584 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Wittig, I., Braun, H.P. & Schagger, H. Blue native PAGE. Nat. Protoc. 1, 418–428 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Reisinger, V. & Eichacker, L.A. Solubilization of membrane protein complexes for blue native PAGE. J. Proteomics 71, 277–283 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Lubec, G., Krapfenbauer, K. & Fountoulakis, M. Proteomics in brain research: potentials and limitations. Prog. Neurobiol. 69, 193–211 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Smith, P.K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

    CAS  Article  Google Scholar 

  28. 28

    Schagger, H., Cramer, W.A. & von Jagow, G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217, 220–230 (1994).

    CAS  Article  Google Scholar 

Download references


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

Author information



Corresponding author

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.