title: Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study.
abstract: BACKGROUND
  Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients.
  
  
  METHODS
  Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics.
  
  
  RESULTS
  The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described.
  
  
  CONCLUSIONS
  We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: K
authors.initials: K
authors.lastname: Smiljanic
authors.affiliation: Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institute and University Hospital, Stockholm, Sweden.
authors.forename: D
authors.initials: D
authors.lastname: Apostolovic
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: S
authors.initials: S
authors.lastname: Trifunovic
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.|Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ognjenovic
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: M
authors.initials: M
authors.lastname: Perusko
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: L
authors.initials: L
authors.lastname: Mihajlovic
authors.affiliation: Institute of Immunology, Virology and Sera Production, Torlak, Belgrade, Serbia.
authors.forename: L
authors.initials: L
authors.lastname: Burazer
authors.affiliation: Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institute and University Hospital, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: van Hage
authors.affiliation: Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.|Ghent University Global Campus, Incheon, South Korea.|Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: Cirkovic Velickovic
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology*; D031164:Ambrosia / Q000276:immunology*; D052179:Antigens, Plant / Q000276:immunology*; D005260:Female; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009245:NADH Dehydrogenase / Q000276:immunology*; D010936:Plant Extracts / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology; D040901:Proteomics; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology; D055815:Young Adult
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D010936:Plant Extracts; D010940:Plant Proteins; C433085:ragweed pollen; D009245:NADH Dehydrogenase
keywords: 
  Ambrosia artemisiifolia
  ; label-free quantification; new short ragweed allergens; pollen allergomes; subpollen particles