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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Kang SU,Fuchs K,Sieghart W,Pollak A,Csaszar E,Lubec Gdoi
10.1038/nprot.2009.92subject
Has Abstractpub_date
2009-01-01 00:00:00pages
1093-102issue
7eissn
1754-2189issn
1750-2799pii
nprot.2009.92journal_volume
4pub_type
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