Fig 1.
Experimental workflow of short ragweed pollen transcriptome, proteome and allergome characterization.
Messenger RNAs from short ragweed pollen were analyzed by deep RNA sequencing using 454 sequencing technology. After de novo transcript assembly, a translated sequence database was generated and used to assign MS/MS spectra from proteomics experiments and perform homology searches. After obtaining a reference 2D-map of the short ragweed pollen proteome, IgE reactivity was analyzed with sera from 22 allergic patients.
Fig 2.
Functional analysis of the short ragweed pollen proteome.
Proteome mining and classification into functional categories was performed using the KAAS server. The histogram denotes numbers of occurrences of KEGG Orthology (KO) annotations (abscissa) assembled in selected categories (ordinate). Among allergens, Amb a 1 and carbonic anhydrase were related to carbohydrate and energy metabolisms, respectively.
Fig 3.
2D-gel reference map of the short ragweed pollen proteome.
Proteins from an aqueous short ragweed pollen extract were separated by 2D-gel electrophoresis and stained with Sypro Ruby. Proteins spots were picked and analyzed by LC-MS/MS after trypsin digestion. Proteins were identified using the Transcriptome-Derived Proteome collection supplemented with missing known allergens. Numbers refer to spots analyzed by mass spectrometry. Identification details are provided in Table 1.
Fig 4.
Mapping of IgE reactivity with short ragweed pollen proteins.
Water soluble short ragweed pollen proteins were resolved by 2D-gel electrophoresis, then probed by western blot with seric IgEs from 22 ragweed-allergic patients. Representative patterns of IgE reactivity obtained from 6 patients (a-f) are shown. Identification details are provided in Table 1.
Table 1.
Assignments of 2D-gel spots containing IgE reactive proteins.
An aqueous short ragweed pollen extract was submitted to 2D-gel electrophoresis followed by western blotting on nitrocellulose membranes with seric IgEs from 22 ragweed-allergic patients. A total of 74 gel plugs (with spot numbers referring to Fig 3) were recovered and assigned after LC-MS/MS analysis using the TDP database. For each spot analyzed, the top 3 master proteins identified in each protein group, based on ≥ 2 peptides sequenced, including ≥ 1 unique one, are listed by decreasing occurences of peptide matches as well as prevalences of IgE reactivity among patients. When several proteins shared the same annotation, a ranking number (#) was assigned.