Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A.
Published
Journal Article
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.
Full Text
Duke Authors
Cited Authors
- Foster, MW; Thompson, JW; Ledford, JG; Dubois, LG; Hollingsworth, JW; Francisco, D; Tanyaratsrisakul, S; Voelker, DR; Kraft, M; Moseley, MA; Foster, WM
Published Date
- August 1, 2014
Published In
Volume / Issue
- 13 / 8
Start / End Page
- 3722 - 3732
PubMed ID
- 25025725
Pubmed Central ID
- 25025725
Electronic International Standard Serial Number (EISSN)
- 1535-3907
Digital Object Identifier (DOI)
- 10.1021/pr500307f
Language
- eng
Conference Location
- United States