MOESM2 of Clinical and immuno-proteomic approach on Lantana camara pollen allergy—a major health hazard

Additional file 2. Snapshots of the MASCOT search for all the peptides. Mascot scores of each peptide is given displaying their homology sequence coverage with other proteins of the other plant species

Mining Novel Allergens from Coconut Pollen Employing Manual <i>De Novo</i> Sequencing and Homology-Driven Proteomics

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual <i>de novo</i> sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish “b” ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its <i>in vitro</i> allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins

Clinical features of the sunflower sensitized patient enrolled in the study.

<p>*Abbreviations: AR- Allergic Rhinitis, BA- Bronchial Asthma, SOB- Shortness of Breath, U- Urticaria, ANG- Angioedema, CC- Cough & Cold, SR- Skin Rash, NS- No Symptom, C1 –C6 –Non-atopic healthy subjects.</p><p>** The grading scale of positive skin prick reactions is as follows: negative if wheal diameter is <3 mm, +1 if wheal diameter is 3–5 mm, +2 if wheal diameter is >6 mm, +3 if wheal diameter is >6 mm, with one or two small pseudopodes, and +4 is any reaction that is more pronounced than +3.</p><p>Clinical features of the sunflower sensitized patient enrolled in the study.</p

BLASTx search analysis using sunflower EST clones (identified in MS/MS) as input against NCBInr database.

<p>#: Query coverage</p><p>##: E value</p><p>###: and sequence identity were calculated for matched proteins in NCBI database with respect to each corresponding EST clones.</p><p>BLASTx search analysis using sunflower EST clones (identified in MS/MS) as input against NCBInr database.</p

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.

<p>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.</p

Search for Allergens from the Pollen Proteome of Sunflower (<i>Helianthus annuus</i> L.): A Major Sensitizer for Respiratory Allergy Patients

<div><p>Background</p><p>Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools.</p><p>Methodology</p><p>Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry.</p><p>Results</p><p>Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease.</p><p>Conclusion</p><p>Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.</p></div

Mass spectrometry based identification of Sunflower (<i>Helianthus annuus</i>) pollen allergens.

<p>*All the scores are as significant as p< 0.05.</p><p>Mass spectrometry based identification of Sunflower (<i>Helianthus annuus</i>) pollen allergens.</p

Total protein profiling and IgE-immunoscreening of allergens from sunflower pollen: A.

<p>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>B</b>. Lane C1-C6: Negative control blot with individual sera of non-atopic (healthy) subjects showing no IgE reactivity</p

List of individual components within allergen class (column clustering) and patient class (row clustering) obtained by Hierarchical Cluster Analysis.

<p>List of individual components within allergen class (column clustering) and patient class (row clustering) obtained by Hierarchical Cluster Analysis.</p

Two dimensional proteome mapping of sunflower pollen and detection of IgE reactive proteins by 2D-immunoblot: A. Proteome of sunflower <i>(H</i>. <i>annuus)</i> 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.

<p>Two dimensional proteome mapping of sunflower pollen and detection of IgE reactive proteins by 2D-immunoblot: A. Proteome of sunflower <i>(H</i>. <i>annuus)</i> 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.</p