PMID: 22338125Feb 18, 2012Paper

Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry.

Molecular & Cellular Proteomics : MCP
Zi-Wei ChenAlex S Evers

Abstract

Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 γ-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the α₁ subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid ...Continue Reading

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