title: Proteomic analysis of wheat proteins recognized by IgE antibodies of allergic patients.
abstract: Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.
authors.affiliation: Department of Immunology and Gnotobiology, Institute of Microbiology v.v.i, Czech Academy of Sciences, Prague, Czech Republic. psotkovsky@gmail.com
authors.forename: Petr
authors.initials: P
authors.lastname: Sotkovský
authors.forename: Martin
authors.initials: M
authors.lastname: Hubálek
authors.forename: Lenka
authors.initials: L
authors.lastname: Hernychová
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Marie
authors.initials: M
authors.lastname: Havranová
authors.forename: Iva
authors.initials: I
authors.lastname: Setinová
authors.forename: Andrea
authors.initials: A
authors.lastname: Kitanovicová
authors.forename: Martin
authors.initials: M
authors.lastname: Fuchs
authors.forename: Jirí
authors.initials: J
authors.lastname: Stulík
authors.forename: Ludmila
authors.initials: L
authors.lastname: Tucková
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology*; D002648:Child; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010940:Plant Proteins / Q000276:immunology / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000009:adverse effects / Q000276:immunology* / Q000378:metabolism; D021182:Wheat Hypersensitivity / Q000175:diagnosis / Q000276:immunology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
chemical_list: D000485:Allergens; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010940:Plant Proteins; D020543:Proteome; D007073:Immunoglobulin E; D000516:alpha-Amylases