A carboxy-terminal affinity tag for the purification and mass spectrometric characterization of integral membrane proteins.
G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.
Wong
Julie P
JP
Biomedical Research Centre, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Reboul
Emmanuelle
E
Molday
Robert S
RS
Kast
Juergen
J
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019713:Receptors, CCR5 / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D019718:Receptors, CXCR4 / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D012243:Rhodopsin / Q000235:genetics; D014162:Transfection
C577836:ABCA1 protein, human; C056475:ABCA4 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; C527276:CXCR4 protein, human; D008565:Membrane Proteins; D009842:Oligopeptides; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin