Mass spectrometric analysis of GABAA receptor subtypes and phosphorylations from mouse hippocampus

Proteomics
Sung Ung KangGert Lubec

Abstract

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 w...Continue Reading

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Citations

Jun 28, 2012·Nature Communications·Jae-Cheol LeeJeung-Whan Han
Mar 3, 2012·Plant & Cell Physiology·Yoichiro Fukao
Feb 10, 2012·Cold Spring Harbor Perspectives in Biology·Dennis van HoofChristine Mummery
Apr 22, 2014·Molecular and Cellular Neurosciences·K Gerrow, A Triller
Feb 22, 2013·Photochemistry and Photobiology·Viktor PajerAntal Nógrádi
Aug 5, 2015·Translational Psychiatry·T M MuellerJ H Meador-Woodruff
Feb 3, 2015·Behavioural Brain Research·Saraswathi SubramaniyanGert Lubec
Jan 29, 2014·Reviews in the Neurosciences·Vincenzo Crunelli, Giuseppe Di Giovanni

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