title: 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-to-protein 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-β-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.
authors.affiliation: From the ‡Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, §Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts xi.zhang.edu@gmail.com.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the ‡Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, §Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Miller
mesh_terms: D002788:Cholesterol Esters / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008320:Maltose / Q000031:analogs & derivatives* / Q000737:chemistry; D008954:Models, Biological; D040901:Proteomics / Q000379:methods; D011963:Receptors, GABA-A / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D002788:Cholesterol Esters; D011963:Receptors, GABA-A; C411362:dodecyl maltopyranoside; D008320:Maltose; C013440:cholesteryl succinate