title: Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.
abstract: Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.
authors.affiliation: †Unit of Glycoconjugate Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), 08034 Barcelona, Spain.
authors.forename: Mònica
authors.initials: M
authors.lastname: Rosa
authors.affiliation: ‡Proteomics Laboratory, Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, ProteoRed ISCIII, 08035 Barcelona, Spain.
authors.forename: Joan Josep
authors.initials: JJ
authors.lastname: Bech-Serra
authors.affiliation: ‡Proteomics Laboratory, Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, ProteoRed ISCIII, 08035 Barcelona, Spain.
authors.forename: Francesc
authors.initials: F
authors.lastname: Canals
authors.affiliation: §Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS), Université de Toulouse, Université Paul Sabatier, 31077 Toulouse, France.
authors.forename: Jean Marie
authors.initials: JM
authors.lastname: Zajac
authors.affiliation: §Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS), Université de Toulouse, Université Paul Sabatier, 31077 Toulouse, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Talmont
authors.affiliation: †Unit of Glycoconjugate Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), 08034 Barcelona, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Arsequell
authors.affiliation: †Unit of Glycoconjugate Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC), 08034 Barcelona, Spain.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Valencia
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D017433:Protein Structure, Secondary; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D002918:Chymotrypsin; D014357:Trypsin
keywords: G-protein coupled receptor (GPCR); MALDI-TOF MS; Orbitrap nanoLC−MS/MS; human Mu-opioid receptor (hMOR); mass spectrometry; membrane protein purification; opioid receptors (ORs); protein structural biology; proteomic analysis; transmembrane protein