Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions Bradley C. Campbell,

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Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions Bradley C. Campbell, PhD, Edward K. Gilding, PhD, Victoria Timbrell, BBMS, Preethi Guru, BSc, Dorothy Loo, MSc, Danila Zennaro, MD, Adriano Mari, MD, Graham Solley, MBBS, Michelle M. Hill, PhD, Ian D. Godwin, PhD, Janet M. Davies, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 1, Pages 133-142 (January 2015) DOI: 10.1016/j.jaci.2014.06.034 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Allergic sensitivity to JGP allergens. A, Correlation between SPT response (millimeters in diameter) to commercial JGP extract and serum IgE to in-house JPG extract. IgE ELISA data are shown as the number of SDs above the mean of nonatopic donors shown in Fig 3. Spearman correlation and 95% CIs are shown. B, IgE immunoblots of JGP probed with sera from patients with grass pollen allergy with symptoms of AR and a positive SPT response to JGP (1-11) and control subjects without pollen allergy (12-19), 2 of whom had house dust mite allergy (15 and 18), and 6 of whom were nonatopic. Molecular weights are shown in kilodaltons. Arrows mark major allergen components. None, No serum control. *Included in the serum pool from patients with JGP allergy in Fig 2. #Included in the serum pool from subjects without pollen allergy. Journal of Allergy and Clinical Immunology 2015 135, 133-142DOI: (10.1016/j.jaci.2014.06.034) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Identification of JGP allergenic components. A, 2D gel electrophoresis of JGP stained with Coomassie Blue. Numbers refer to spots excised for proteomics. 2D IgE immunoblots of JGP probed with a serum pool from patients with JGP allergy. B, Patients from Fig 1. Arrows mark IgE-reactive components. A replica immunoblot with a pool of sera from nonatopic subjects from Fig 1 showed no IgE reactivity (data not shown). C and D, Specific mAb. Sor h 1 and Sor h 13 isoforms are marked. Journal of Allergy and Clinical Immunology 2015 135, 133-142DOI: (10.1016/j.jaci.2014.06.034) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Serum IgE reactivity with Sor h 1 and Sor h 13. A, Purified JGP allergens stained with Coomassie blue and immunoblotted with mAb specific to group 1 and group 13 allergens (marked). B and C, IgE reactivity normalized to nonatopic (NA) donors. Box, Median and interquartile range; whiskers, 10th to 90th percentiles. The cutoff of 3 SDs above the mean of 23 nonatopic subjects is shown for JGP (OD = 0.410) and Sor h 1 (OD = 0.229) in Fig 3, B, and for JGP (OD = 0.371) and Sor h 13 (OD = 0.384) in Fig 3, C. P values were determined by using the Mann-Whitney U test. D and E, Spearman (with 95% CIs) correlation for IgE reactivity with JGP and Sor h 1 (Fig 3, D) and Sor h 13 (Fig 3, E). F, Frequency of IgE reactivity with JGP, Sor h 1, and Sor h 13. GP, Patients with grass pollen allergy; OA, subjects with allergies other than grass pollen. Journal of Allergy and Clinical Immunology 2015 135, 133-142DOI: (10.1016/j.jaci.2014.06.034) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Analysis pipeline for the total JGP proteome, transcriptome, and allergome. Data file names of raw results and summaries for each step toward deducing the actual IgE-reactive allergen components and putative allergens of JGP are given. The number of entries in each data set are specified. ms/ms, Tandem mass spectrometry; NCBI, National Center for Biotechnology Information; NR, nonredundant plant database; seq, sequence. Journal of Allergy and Clinical Immunology 2015 135, 133-142DOI: (10.1016/j.jaci.2014.06.034) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions