title: Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A.
abstract: Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC-MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Foster
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.forename: Julie G
authors.initials: JG
authors.lastname: Ledford
authors.forename: Laura G
authors.initials: LG
authors.lastname: Dubois
authors.forename: John W
authors.initials: JW
authors.lastname: Hollingsworth
authors.forename: Dave
authors.initials: D
authors.lastname: Francisco
authors.affiliation: ‡Department of Medicine, National Jewish Health, Denver, Colorado 80206, United States.
authors.forename: Sasipa
authors.initials: S
authors.lastname: Tanyaratsrisakul
authors.affiliation: ‡Department of Medicine, National Jewish Health, Denver, Colorado 80206, United States.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Voelker
authors.forename: Monica
authors.initials: M
authors.lastname: Kraft
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: W Michael
authors.initials: WM
authors.lastname: Foster
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D018893:Bronchoalveolar Lavage; D001992:Bronchoalveolar Lavage Fluid; D002853:Chromatography, Liquid; D014644:Genetic Variation; D005838:Genotype; D064368:Healthy Volunteers; D006579:Heterozygote; D006801:Humans; D008168:Lung / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000737:chemistry; D040901:Proteomics; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D055815:Young Adult
chemical_list: D010455:Peptides; D020033:Protein Isoforms; D037662:Pulmonary Surfactant-Associated Protein A; C445842:SFTPA1 protein, human; C445843:SFTPA2 protein, human; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin
keywords: allelic variant; ionKey; rs1965708; surfactant-associated protein; targeted proteomics