Table 1.
Clinical features of the sunflower sensitized patient enrolled in the study.
Fig 1.
Total protein profiling and IgE-immunoscreening of allergens from sunflower pollen: A.
Lane M: Medium range molecular weight marker, Lane T: Total protein profile of sunflower pollen in 12% SDS-PAGE as visualized by staining with CBB-R250, Lane 1–20: immunoblotting with individual serum of twenty sunflower positive atopic patients, Lane C: Negative control blot with pooled sera of non-atopic (healthy)subjects showing no IgE reactivity; B. Lane C1-C6: Negative control blot with individual sera of non-atopic (healthy) subjects showing no IgE reactivity
Fig 2.
Two dimensional proteome mapping of sunflower pollen and detection of IgE reactive proteins by 2D-immunoblot: A. Proteome of sunflower (H. annuus) pollen in 2D gel within a pH range 3–10; B. 2D immunoblot with pooled sera from 20 atopic patients revealed seven distinct IgE reactive spots on 2D blot.
Fig 3.
Detection of glycoprotein allergens in sunflower pollen and their antigenecity by metaperiodate mediated deglycosylation study in 2D blot: A. PAS staining of sunflower pollen protein in 1D SDS-PAGE to detect the glycoproteins, which turned into purple colors. The PAS stained gel was compared with IgE-immunoblot shown in Fig 1, which identified three glycoprotein regions as possible IgE reactive zones highlighted in boxes. Lane 1: sunflower pollen rotein in SDS-PAGE after PAS staining, Lane 2: Counter staining the PAS stained gel with CBB- G250 that turned only non-glycoproteins into blue. B & C. 2D immunoblot without metaperiodate treatment (B) was compared with another 2D immunoblot after metaperiodate (C) treatment.
The metaperiodate treatment resulted in loss of IgE reactivity of certain spots (marked within box) suggesting the possible involvement of sugar moiety to determine the IgE binding of the allergens.
Fig 4.
Hierarchical cluster analysis based on individual patient’s immunological profile and the corresponding immuno-reactive components: A. Heat map representing a qualitative summary matrix based on presence/absence of IgE-reactive components in 2D immunoblots of individual patient serum. The clustered data was processed with complete linkage according to Euclidean distance. Column clustering divided allergens into four classes (A- D) based on their frequency of IgE-reactivity. Row clustering divided patients into four classes (1–4) depending upon the sensitivity towards sunflower pollen allergen. B. Scatter plot analysis of sunflower specific IgE levels in serum pool of each of the four patient clusters generated by hierarchical cluster analysis.
Each dot represents a single patient within a cluster.
Table 2.
List of individual components within allergen class (column clustering) and patient class (row clustering) obtained by Hierarchical Cluster Analysis.
Fig 5.
A schematic representation illustrating the mass spectrometry workflow adopted in the present study for proteomic identification of sunflower.
Table 3.
Mass spectrometry based identification of Sunflower (Helianthus annuus) pollen allergens.
Table 4.
BLASTx search analysis using sunflower EST clones (identified in MS/MS) as input against NCBInr database.