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 replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 γ-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the α₁ subunit, confirmed two splice variants of the γ₂ subunit (γ₂L and γ₂S) and resolved a database discrepancy in the sequence of the α₅ subunit.

Fig. 1.

SDS-PAGE of immunopurified GABA_A receptors. A, Immunoblot with antibody to the α₁ subunit of the GABA_A receptor. B, SYPRO-Ruby™ protein staining. C, Peptide level sequence coverage of the α₁ subunit of the GABA_A receptor based on MS2 spectra obtained from nano-LC-MS analysis of the in-gel tryptic digest of band 3 from panel B. The red highlights represent regions with MS2 identified peptide sequences.

Fig. 2.

Time course of chymotryptic digestion to obtain increased sequence coverage of the fourth transmembrane domain of the GABA_A receptor α₁ subunit using LC-MS. A, Diagram of a GABA_A receptor subunit composed of four transmembrane spanning regions with extracellular N- and C termini and the assembly of five such subunits to form a pentameric chloride channel. B, Overlapping peptides of the entire fourth TMD of the α₁ subunit of the GABA_A receptor from the sequential digestion with Lys-C and chymotrypsin for varying lengths of time. C, Peptide intensities from MS1 spectra of the indicated TMD4 peptides that were produced during the chymotryptic digestion time course.

Fig. 3.

Multi-enzyme digestion of the N-terminal region of the α₁ subunit of the GABA_A receptor. (Top) Overlapping peptides from tryptic and chymotryptic digestions. A, MS2 spectrum of a Lys-C/tryptic peptide, ²¹LLDGYDNR²⁸. B, MS2 spectrum of a Lys-C/chymotryptic peptide, ¹⁵TRILDRLLDGY²⁵.

Fig. 4.

Identification of deglycosylated N-terminal peptides from the α₁ subunit of the GABA_A receptor following treatment with PNGase F. A, Western blot with an anti-α₁ antibody comparing untreated and PNGase-treated receptors. B, The MS2 spectrum of a tryptic peptide corresponding to ¹QPSQDELKDDTTVFTR¹⁶ (m/z = 931.936, z = 2). The N-terminal Q was modified to pyro-E, labeled as pE. C, The MS2 spectrum of a tryptic peptide corresponding to ¹⁰⁶SVAHDMTM^oxidationKPNK¹¹⁶ (m/z = 623.779, z = 2). The N to D change produced by PNGase F catalyzed deamidation is denoted by the gray D and arrow.

Fig. 5.

Comparison of total and TMD peptides from the α₁ subunit of the GABA_A receptor recovered by sequential solid phase extraction with C4 and porous graphitic carbon. A, Peptides recovered using C4 and PGC SPE from the total peptide pool (top); peptide size distribution of the total peptides recovered by C4 and PGC SPE (lower). B, TMD peptides recovered by C4 and PGC SPE (top); size distribution of TMD peptides recovered by C4 and PGC SPE (lower). Bars indicate mean ± S.E.

Fig. 6.

High resolution mass spectrometry improves the identification of peptides in the fragmentation spectra (MS2) of the tryptic peptide ¹⁸⁷LNQYDLLGQTVDSGIVQSSTGEYVVM^oxidationTTHFHLK²¹⁹ (m/z = 924.962⁺⁴). Signals from peptide fragmentation are assigned using both enhanced mass accuracy and charge state determination. Accurate charge state assignment from isotopic resolution is illustrated in bottom spectra.

Fig. 7.

Residue level sequencing of the α₁ subunit of the GABA_A receptor, purified from rat brain neocortex. Amino acid residues definitively identified from analysis of fragment ions of the MS2 spectra are in red and BOLD. The residue coverage of the whole protein is 80%. Frames indicate the TMDs of the protein. The residue coverage of the TMDs is 63%. The sequencing coverage of this protein at the peptide level is 96%. The missing peptides/residues are ⁷⁴LK⁷⁵, ¹³²L, ²⁷⁴N, ²³¹LPCIM²³⁵, ²⁸⁹IAVCY²⁹³, and ³¹⁰TK³¹¹. ²⁸⁹IAVCY²⁹³ was observed in both new replicates.

Fig. 8.

High-resolution LC-MS workflow for sequencing picomole quantities of transmembrane proteins from biological samples. Pentameric GABA_A receptors were purified from an immunoaffinity column. The proteins were denatured and delipidated followed by timed, in-solution digestion with several proteolytic enzymes. Peptide recovery was enhanced using sequential solid phase extraction. Peptide identification and sequencing was optimized using nano-LC-ESI-MS with high resolution in both MS1 and MS2 spectra.

Fig. 9.

Venn diagram showing overlapping amino acid sequence coverage of the α₁ subunit from three replicate brain samples. Peptides covering 392 (Replicate A; original sample), 353 (Replicate B), and 378 (Replicate C) amino acids were identified, representing 97%, 88%, and 94% of the detectable amino acid sequence of the α₁ subunit.

Fig. 10.

Relative abundance of GABA_A receptor subunits determined by label-free quantitative analysis. The mean ± S.E. of the normalized spectrum counts for three replicate samples are shown on the graph, with the mean value labeled for each subunit. The stoichiometry of α: β: (γ+δ) = 2.3: 2.4: 1, consistent with the expected 2: 2: 1 stoichiometry.

Fig. 11.

Identification of γ₂ subunit splice variant isoforms of the GABA_A receptor. A, The γ_2S and γ_2L splice variants differ by 8 amino acids (LLRMFSF; underlined). B, An MS2 spectrum of the tryptic peptide, NPAPTIDIRPR (m/z = 625.354²⁺) was observed, identifying the γ_2S variant isoform. C, An MS2 spectrum of the chymotryptic peptide KNAPTIDIRPR (m/z = 583.853²⁺) was observed, identifying the γ_2L variant isoform.

Fig. 12.

Resolution of a single amino acid database discrepancy in the sequence of the α₅ subunit. A, Amino acid differences in residue 168 of the α₅ subunit are indicated by the arrow. B, The MS2 spectrum of a peptide (m/z = 936.091³⁺) consistent with ¹⁶⁷LTISAEC*PmQLEDFPmDAHAC*PLK¹⁹⁰ (theoretical m/z = 936.090³⁺, * indicates carbamidomethylation of Cys) was observed, suggesting that ¹⁶⁸Thr is the correct residue in the α₅ subunit from our biological source.