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A Carboxy-Terminal Affinity Tag for the Purification and Mass Spectrometric Characterization of Integral Membrane Proteins

TitleA Carboxy-Terminal Affinity Tag for the Purification and Mass Spectrometric Characterization of Integral Membrane Proteins
Publication TypeJournal Article
Year of Publication2009
AuthorsWong, JP, Reboul, E, Molday, RS, Kast, J
JournalJournal of Proteome Research
Date PublishedMay
Type of ArticleArticle
ISBN Number1535-3893
Keywords1D4 affinity tag, 2-DIMENSIONAL, annotated spectrum libraries, ATP binding cassette transporters, BINDING, ELECTROPHORESIS, EXPRESSION, G-protein-coupled, membrane protein enrichment, N-LINKED GLYCOSYLATION, PROTEOMICS, proteotypic peptides, RECEPTOR, RECEPTORS, RHODOPSIN, ROD OUTER SEGMENTS, SITES, SYNTHETIC PEPTIDES, Tandem mass spectrometry

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.

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