title: Development of structural marker peptides for cystic fibrosis transmembrane conductance regulator in cell plasma membrane by reversed-footprinting mass spectrometry.
abstract: A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment. In searching for structurally informative peptides, a workflow was designed for accurate and precise quantitation of CFTR peptides produced from proteolytically digesting the plasma membrane subproteome of cells. This sample preparation strategy mitigated the need for challenging purification of large quantities of structurally intact CFTR. On the basis of the interrogated peptides, it was proposed a concept of the structural marker peptide that could report CFTR structure and function in cells. The reversed-footprinting mass spectrometry extends the FPOP technology to study conformation and interaction changes of low-abundance proteins directly in their endogenous cellular locations.
authors.forename: Vahid
authors.initials: V
authors.lastname: Farrokhi
authors.forename: Bekim
authors.initials: B
authors.lastname: Bajrami
authors.forename: Reza
authors.initials: R
authors.lastname: Nemati
authors.forename: Adam J
authors.initials: AJ
authors.lastname: McShane
authors.affiliation: Department of Physics, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Franz
authors.initials: F
authors.lastname: Rueckert
authors.affiliation: Department of Physics, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Barrett
authors.initials: B
authors.lastname: Wells
authors.forename: Xudong
authors.initials: X
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D015415:Biomarkers / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D023302:Protein Footprinting
chemical_list: D015415:Biomarkers; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator