Mass spectrometric analysis of GABA A receptor subtypes and phosphorylations from mouse hippocampus

SungUng Kang, Seok Heo, Gert Lubec

Research output: Contribution to journalArticle

Abstract

The brain GABA A receptor (GABA AR) is a key element of signaling and neural transmission in health and disease. Recently, complete sequence analysis of the recombinant GABA AR 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, 1.5M aminocaproic acid in the presence of 0.3M 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 60kDa 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 AR-specific MS information taken from own previous studies was used for searches of GABA AR 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 AR 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 AR studies at the protein chemical rather than at the immunochemical level in health and disease.

Original languageEnglish (US)
Pages (from-to)2171-2181
Number of pages11
JournalProteomics
Volume11
Issue number11
DOIs
StatePublished - Jun 2011
Externally publishedYes

Fingerprint

Phosphorylation
GABA-A Receptors
Hippocampus
Gels
Health
Aminocaproic Acid
Native Polyacrylamide Gel Electrophoresis
Subtilisin
Octoxynol
Phosphoric Monoester Hydrolases
Synaptic Transmission
Health Status
Sequence Analysis
Sucrose
Brain
Membrane Proteins
Proteins
Molecular Weight
Molecular weight
Ions

Keywords

  • Animal proteomics
  • GABA receptor
  • MS
  • Post-translational modifications

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Mass spectrometric analysis of GABA A receptor subtypes and phosphorylations from mouse hippocampus. / Kang, SungUng; Heo, Seok; Lubec, Gert.

In: Proteomics, Vol. 11, No. 11, 06.2011, p. 2171-2181.

Research output: Contribution to journalArticle

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