Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

Abstract

The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-toprotein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/ peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-␤-D-maltopyranoside/cholesteryl hemisuccinate (DDM/ CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses-without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. Molecular & Cellular Proteomics 14: 10.1074/ mcp.M114.042556, 724-738, 2015. Transmembrane (TM)1 proteins are abundant and play critical roles in nearly all biological processes. About 25% of the 29,375 unique protein sequences in human proteome contain at least one TM alpha-helix, and 13% contain at least two (1). TM proteins such as ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs) are cell gate-keepers that convert external signals into cellular activities throughout the central nervous system (CNS), and predominate as desired drug targets. Ion channels already represent 10% of current drug targets before the structure-function mechanisms of CNS LGICs become clear (1). Formed by five homologous TM subunits (main form in brain (␣1) 2 (␤2/␤3) 2 (␥2L) 1 ) (2, 3), Cysloop LGIC gamma-aminobutyric acid type A receptor (GABA A R) is the major inhibitory (Cl) ion channel that balances excitation in CNS. Genomic studies have pinpointed several single mutations in GABA A R as the causes-and potential drug targets-of epilepsy, a devastating disorder that afflicts 2.2 million Americans and 65 million people worldwide at tremendous health and economic costs (4 -12). For decades, antiepileptic drugs remain limited, control only 2/3 of epilepsies, and incur serious side effects including sedation, addiction, dizziness, and suicidality, indicating urgency for more effective mechanism-driven pharmacotherapy. However, no atomic stru...

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

“…HCD settings were: precursor ion isolation width 2.0 Da for the quadrupole (Q) mass filter and normalized collision energy 25% for the highpressure collision cell. Dynamic exclusion of precursor ions was applied with a 0.2 Da width for durations of 20 s. For the few high-low (H-L) HPLC MS and CID MS/MS experiments on LTQ-Orbitrap XL using top10 method, data acquisition settings were the same as described earlier (34). MS/MS acquisition included singly charged precursors to strengthen peptide redundancy desired in HDX and pan-PTM mapping, to cover nonpeptide endogenous components (16), and to aid method evaluation and diagnose in this pilot platform development.…”

“…The membranes were solubilized by gradually adding 20 mM (1%, m/v) DDM/4 mM (0.25%, m/v, Trizma salt, equivalent to 0.2% free acid form) CHS over 2 mg/ml protein; solubilized supernatant was loaded to an anti-FLAG column operated at constant 0.3 ml/min for efficiency and consistency; GABA A R was eluted with FLAG peptide, cleaned and concentrated using dial-filtration with a 100 kDa molecular weight cutoff (MWCO) filter to 25 M (BCA assay, using pentamer molecular weight 285 kDa), in the protein buffer composed of 1 mM (0.05% m/v) DDM, 0.2 mM (0.0125% m/v) CHS, 10% (v/v) glycerol, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM CaCl 2 , 5 mM KCl, 5 mM MgCl 2 , and 4 mM EDTA. Purified protein's receptor activity, state of dispersion (nonaggregation), and protein composition were validated as described earlier (34). Biological variances were covered by pooling 120 cell plates from two batches of growth at different passage number, and technical variances were addressed by a repeated split-pool strategy (34).…”

“…Purified protein's receptor activity, state of dispersion (nonaggregation), and protein composition were validated as described earlier (34). Biological variances were covered by pooling 120 cell plates from two batches of growth at different passage number, and technical variances were addressed by a repeated split-pool strategy (34).…”

“…GABA A R Protein Purification from HEK293 Cells-HEK293-TetR cells stably over-expressing full-length human (N)-FLAG-␣1␤3␥2L-(GGS) 3 GK-1D4 GABA A R at ␣1 (bovine): ␤3 isoform 2 (human): ␥2L (human) subunit plasmid copy ratio of 2:2:1 were provided by Dr. Keith W. Miller (35), and final active GABA A R (at typical concentration of 25 M in the 0.05% DDM/0.0125% CHS/10% glycerol protein buffer) was obtained by affinity purification on a low-flow-rate anti-FLAG column, as described earlier (34). Briefly, following 72-hour growth and 24-hour tetracycline-induced GABA A R expression in 5% CO 2 at 37°C, cells at 90% confluence on 15-cm plates were PBSwashed, harvested into hypotonic lysis buffer (10 mM HEPES pH 7.5, protease inhibitors LACP and PMSF), and frozen slowly at Ϫ80°C to promote lysis.…”

“…Pepsin digests were searched without enzyme specification, and porcine pepsin was added to the sequence database for test. Earlier high-low CID MS/MS data were searched with mass tolerance 10 ppm and 0.8 Da (or 0.2 Da when specified) for MS and MS/MS respectively, same as described (34).…”