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Mass spectrometric analysis of GABA A receptor subtypes and phosphorylations from mouse hippocampus
Author(s) -
Kang Sung Ung,
Heo Seok,
Lubec Gert
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000374
Subject(s) - trypsin , chemistry , chromatography , coomassie brilliant blue , phosphatase , subtilisin , biochemistry , protein subunit , biology , phosphorylation , enzyme , staining , gene , genetics
The brain GABA A receptor (GABA A R) is a key element of signaling and neural transmission in health and disease. Recently, complete sequence analysis of the recombinant GABA A R has been reported, separation and mass spectrometrical (MS) characterisation from tissue, however, has not been published so far. Hippocampi were homogenised, put on a sucrose gradient 10–69% and the layer from 10 to 20% was used for extraction of membrane proteins by a solution of Triton X‐100, 1.5 M aminocaproic acid in the presence of 0.3 M Bis‐Tris. This mixture was subsequently loaded onto blue native PAGE (BN‐PAGE) with subsequent analysis on denaturing gel systems. Spots from the 3‐DE electrophoretic run were stained with Colloidal Coomassie Brilliant Blue, and spots with an apparent molecular weight between 40 and 60 kDa were picked and in‐gel digested with trypsin, chymotrypsin and subtilisin. The resulting peptides were analysed by nano‐LC‐ESI‐MS/MS (ion trap) and protein identification was carried out using MASCOT searches. In addition, known GABA A R‐specific MS information taken from own previous studies was used for searches of GABA A R subunits. β‐1, β‐2 and β‐3, θ and ρ‐1 subunits were detected and six novel phosphorylation sites were observed and verified by phosphatase treatment. The method used herein enables identification of several GABA A R subunits from mouse hippocampus along with phosphorylations of β‐1 (T227, Y230), β‐2 (Y215, T439) and β‐3 (T282, S406) subunits. The procedure forms the basis for GABA A R studies at the protein chemical rather than at the immunochemical level in health and disease.