title: Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications.
abstract: Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.
authors: Gregory Fleming James Cystic Fibrosis Research Center,  University of Alabama at Birmingham, Birmingham, AL 35294,  USA.
authors: Michelle
authors: M
authors: McClure
authors: Lawrence J
authors: LJ
authors: DeLucas
authors: Landon
authors: L
authors: Wilson
authors: Marjorie
authors: M
authors: Ray
authors: Steven M
authors: SM
authors: Rowe
authors: Xiaoyun
authors: X
authors: Wu
authors: Qun
authors: Q
authors: Dai
authors: Jeong S
authors: JS
authors: Hong
authors: Eric J
authors: EJ
authors: Sorscher
authors: John C
authors: JC
authors: Kappes
authors: Stephen
authors: S
authors: Barnes
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012694:Serine