Complete sequencing of GABAA receptor subunit beta 3 by a rapid technique following in-gel digestion of the protein.
Knowledge on complete sequences is pivotal to identify splice variants, generate specific antibodies and predict conformation. A simple analytical approach to obtain 100% sequence coverage, however, is currently not available. Recombinant gamma-aminobutyric acid A receptor subunits were from insect SF9 cells that were co-transfected with rat alpha1 and His-tag beta 3. The complex of these two subunits was run on blue-native PAGE, followed by multidimensional electrophoretic steps. Spots resolved at the third electrophoretic step were in-gel digested with trypsin, chymotrypsin and Asp-N. In-gel modification of lysines by acetylation was carried out to increase sequence coverage. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using both, collision-induced dissociation and electron transfer dissociation principles. When results from trypsin, chymotrypsin and Asp-N digestion were combined, a single peptide [424 KKTHLRRRSSQL 435] was still not identified. In-gel lysine acetylation leads to unambiguous identification of this peptide by the use of MASCOT v2.2. The use of the Modiro software along with MASCOT, however, was able to provide 100% sequence coverage even without the use of in-gel lysine acetylation. It was observed that the use of trypsin, chymotrypsin and Asp-N with bioinformatic handling by the MASCOT and Modiro software is sufficient to obtain complete sequencing of a highly hydrophobic membrane protein.
Kang
Sung Ung
SU
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
Lubec
Gert
G
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D021122:Protein Subunits; D040901:Proteomics / Q000379:methods; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D010446:Peptide Fragments; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D013388:Succinimides; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D008239:Lysine