title: Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines.
abstract: Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety. Proteomics has become an invaluable tool for characterization and quantification of the protein complement of cereal grains. In this study multiple reaction monitoring (MRM) mass spectrometry, viewed as the gold standard for peptide quantification, was compared to the data-independent acquisition strategy known as SWATH-MS (sequential window acquisition of all theoretical mass spectra). SWATH-MS was comparable (p < 0.001) to MRM-MS for 32/33 peptides assessed across the four families of hordeins (gluten) in eight barley lines. The results of SWATH-MS analysis further confirmed the absence of the B-, C-, and D-hordeins in the triple-null barley line and showed significantly reduced levels ranging from <1% to 16% relative to wild-type (WT) cv Sloop for the minor γ-hordein class. SWATH-MS represents a valuable tool for quantitative proteomics based on its ability to generate reproducible data comparable with MRM-MS, but has the added benefits of allowing reinterrogation of data to improve analytical performance, ask new questions, and in this case perform quantification of trypsin-resistant proteins (C-hordeins) through analysis of their semi- or nontryptic fragments.
authors.affiliation: CSIRO Agriculture, 306 Carmody Road, St Lucia, Queensland 4067, Australia.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Colgrave
authors.affiliation: CSIRO Agriculture, 306 Carmody Road, St Lucia, Queensland 4067, Australia.
authors.forename: Keren
authors.initials: K
authors.lastname: Byrne
authors.affiliation: CSIRO Agriculture, GPO Box 1600, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Blundell
authors.affiliation: SCIEX, Phoenix House, Lakeside Drive, Centre Park, Warrington, WA1 1RX, U.K.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Heidelberger
authors.affiliation: SCIEX, Phoenix House, Lakeside Drive, Centre Park, Warrington, WA1 1RX, U.K.
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Lane
authors.affiliation: CSIRO Agriculture, GPO Box 1600, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Tanner
authors.affiliation: CSIRO Agriculture, GPO Box 1600, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Crispin A
authors.initials: CA
authors.lastname: Howitt
mesh_terms: D002446:Celiac Disease / Q000178:diet therapy; D005983:Glutens / Q000032:analysis* / Q000235:genetics; D001467:Hordeum / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009154:Mutation; D010455:Peptides / Q000032:analysis / Q000235:genetics; D000069600:Plant Breeding; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides; D010940:Plant Proteins; D005983:Glutens