Deep Amino Acid Sequencing of Native Brain GABAA Receptors Using High-Resolution Mass Spectrometry

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 phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α1 subunit was observed in all three with in of the three a high of sequence coverage was to the three to the of γ-aminobutyric acid type A receptor The sequence on the α1 subunit, of the subunit and and a database in the sequence of the 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 phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α1 subunit was observed in all three with in of the three a high of sequence coverage was to the three to the of γ-aminobutyric acid type A receptor The sequence on the α1 subunit, of the subunit and and a database in the sequence of the Mass spectrometric sequencing of from a for by mass using a of the and in protein sequence from and and to to the of protein from samples of the sequence and of sequence the amino acid residue sequence from of database to the and of amino acid and and sequence The of residue mass spectrometric sequencing to integral membrane membrane graphitic phase