title: Using LC-MS Based Methods for Testing the Digestibility of a Nonpurified Transgenic Membrane Protein in Simulated Gastric Fluid.
abstract: The digestibility of a nonpurified transgenic membrane protein was determined in pepsin, as part of the food safety evaluation of its resistance to digestion and allergenic potential. Delta-6-desaturase from Saprolegnia diclina, a transmembrane protein expressed in safflower for the production of gamma linolenic acid in the seed, could not be obtained in a pure, native form as normally required for this assay. As a novel approach, the endoplasmic reticulum isolated from immature seeds was digested in simulated gastric fluid (SGF) and the degradation of delta-6-desaturase was selectively followed by SDS-PAGE and targeted LC-MS/MS quantification using stable isotope-labeled peptides as internal standards. The digestion of delta-6-desaturase by SGF was shown to be both rapid and complete. Less than 10% of the initial amount of D6D remained intact after 30 s, and no fragments large enough (>3 kDa) to elicit a type I allergenic response remained after 60 min.
authors.affiliation: Arcadia Biosciences , 202 Cousteau Place, Suite 200, Davis, California 95618, United States.
authors.forename: Wayne S
authors.initials: WS
authors.lastname: Skinner
authors.affiliation: Proteomics Core Facility, University of California , Room 1414 GBSF, 451 East Health Sciences Drive, Davis, California 95616, United States.
authors.forename: Brett S
authors.initials: BS
authors.lastname: Phinney
authors.affiliation: Proteomics Core Facility, University of California , Room 1414 GBSF, 451 East Health Sciences Drive, Davis, California 95616, United States.
authors.forename: Anthony
authors.initials: A
authors.lastname: Herren
authors.affiliation: Arcadia Biosciences , 202 Cousteau Place, Suite 200, Davis, California 95618, United States.
authors.forename: Floyd J
authors.initials: FJ
authors.lastname: Goodstal
authors.affiliation: Arcadia Biosciences , 202 Cousteau Place, Suite 200, Davis, California 95618, United States.
authors.forename: Isabel
authors.initials: I
authors.lastname: Dicely
authors.affiliation: Arcadia Biosciences , 202 Cousteau Place, Suite 200, Davis, California 95618, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Facciotti
mesh_terms: D000595:Amino Acid Sequence; D031187:Carthamus tinctorius / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D004063:Digestion; D030841:Food, Genetically Modified; D005750:Gastric Juice / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D008565:Membrane Proteins; D010940:Plant Proteins
keywords: LC-MS; food safety evaluation; genetically engineered plants; mass spectrometry; membrane proteins; protein digestibility