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Induced sputum proteome in healthy subjects and asthmatic patients

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1 Induced sputum proteome in healthy subjects and asthmatic patients
Sina A. Gharib, MD, Elizabeth V. Nguyen, BS, Ying Lai, PhD, Jessica D. Plampin, BS, David R. Goodlett, PhD, Teal S. Hallstrand, MD, MPH  Journal of Allergy and Clinical Immunology  Volume 128, Issue 6, Pages e6 (December 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Pulmonary function profiles of subjects. FEV1 values of 10 asthmatic patients (5 EIB+ and 5 EIB− subjects) and 5 healthy control subjects (NC) are shown as baseline (Pre) and temporally after exercise challenge. Note that the asthmatic group has a lower baseline FEV1, but only the EIB+ subjects have further airflow obstruction after exercise. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Functional analysis of the induced sputum proteome. A, Enriched biological modules representing biological processes, molecular functions, and cellular components are depicted based on the hierarchical structure of GO annotation. B, The same functional categories are rank ordered based on their enrichment P values. HDL, High-density lipoprotein. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Protein interaction network of induced sputum. This interactome is a subset of the entire induced sputum proteome and is characterized by experimentally verified functional interaction of its member gene products. Selected proteins upregulated (red) and downregulated (green) in asthma are highlighted, as are 2 proteins upregulated in EIB+ subjects in response to exercise challenge (magenta). Note the interaction of the downregulated protein SCGB1A with phospholipase A2 and the high connectivity of the C3 hub (please see text for discussion). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Proteomic signature of induced sputum in asthmatic patients. A, Hierarchical clustering of differentially expressed proteins based on spectral counting is represented as a heat map and demonstrates robust discrimination between asthmatic patients and control subjects. B-D, Confirmation of proteomics findings for select proteins using Western blot analysis. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Exercise-induced alterations of the induced sputum proteome in asthmatic patients with EIB. A, Heat map depiction of differentially expressed proteins in EIB+ subjects on exercise challenge. B and C, Immunoblot analysis confirms the shotgun proteomics results for the upregulation of C3a in response to exercise and trends toward significance for HPX. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Cellular differential of induced sputum obtained at baseline (top panel) and after exercise challenge (bottom panel). No statistically significant differences between the subjects at baseline versus after exercise were observed. Eos, Eosinophils; Epi, epithelial cells; Lym, lymphocytes; Mac, macrophages; NC, healthy control subjects; PMN, polymorphonuclear cells. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 Principal components analysis of the induced sputum proteome. PCA is a method to reduce the complexity of data structures in such a way as to capture most of its variability. The 3 axes shown in the figure correspond to the 3 principal components that capture the majority of variability in protein expression across all subjects. Each sphere corresponds to an individual subject. When PCA is applied to the set of all identified proteins in induced sputum, no clear clustering of subjects based on their phenotype is observed, implying that the presence of asthma does not appear to perturb the global variability of induced sputum protein expression. EIB+, Asthmatic patients with EIB; EIB−, asthmatic patients without EIB. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Western analysis of selected proteins in asthmatic patients with and without EIB and control subjects. Although the expression levels of these proteins were significantly different between asthmatic patients versus control subjects, no significant differences were observed between asthma subphenotypes (EIB+ vs EIB−). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Hierarchical clustering of differentially expressed proteins between asthmatic subjects with and without EIB represented as a heat map. A2M, α2-macroglobulin; FGB, fibrinogen β chain; GSTP1, glutathione-S-transferase π 1; HBB, hemoglobin β; HIST1H4, histone cluster 1 H4. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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