Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease Maxie Kohler, PhD, AnnSofi Sandberg,

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Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease Maxie Kohler, PhD, AnnSofi Sandberg, MSc, Sanela Kjellqvist, PhD, Andreas Thomas, PhD, Reza Karimi, MD, Sven Nyrén, MD, PhD, Anders Eklund, MD, PhD, Mario Thevis, PhD, C. Magnus Sköld, MD, PhD, Åsa M. Wheelock, PhD Journal of Allergy and Clinical Immunology Volume 131, Issue 3, Pages 743-751.e9 (March 2013) DOI: 10.1016/j.jaci.2012.09.024 Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Optimized models showing markers for smokers versus patients with COPD (R2 = 0.50, Q2 = 0.45; A), female smokers versus female patients with COPD (R2 = 0.81, Q2 = 0.78; B), male smokers versus male patients with COPD (R2 = 0.49, Q2 = 0.42; C), and male patients with COPD versus female patients with COPD (R2 = 0.76, Q2 = 0.71; D). Left panels show score plots (Fig 1, A-C: gray, smokers; black, patients with COPD; Fig 1, D: gray, male patients; black, female patients), with the predictive component along the y-axes. Because no orthogonal components were required, the x-axes merely represent a numeric ordering (Num) of the samples. Right panels display the VIP. Arrows indicate upregulation or downregulation in patients with COPD compared with that seen in healthy smokers (Fig 1, A-C) or regulation in female compared with male patients (Fig 1, D). Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Analysis of shared and unique structures comparing OPLS gender-specific models for healthy smokers versus smokers with COPD for male (x-axis) and female (y-axis) gender. In contrast to Fig 1, the models based on all variables are used here (see Table II for model statistics). Marked variables are most important for differentiation of male (italic) or female (bold) smokers versus patients with COPD. Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Analysis of shared and unique structures between OPLS models of patients with COPD versus healthy subjects for smoking (x-axis) and nonsmoking (y-axis) populations. Clustering around the diagonal indicates that the same proteins are altered because of COPD, regardless of smoking. Marked spots are the most important variables for separating smokers and smoking patients with COPD (Fig 1). Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Validation of 2D-DIGE results by means of Western blotting. A, Cathepsin B; B, lamin A/C. Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Representative gel image of the 2DE-separated soluble proteins from BAL cells of a pool of healthy volunteers, smokers, and patients with COPD (internal standard). The spots indicated are further described in Fig 2 and Tables III and E2 and represent the most important findings in this study. Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 SUS plot comparing the predictive component of the models comparing all female smokers with female patients with COPD (x-axis; R2 = 0.94, Q2 = 0.70) and female postmenopausal smokers versus female postmenopausal patients with COPD (y-axis; R2 = 0.97, Q2 = 0.72). As evident from the diagonal distribution of the variables, menopause does not appear to cause any substantial alterations to the variable contribution. The protein spots that are most affected by the removal of premenopausal women from the model (furthest away from the diagonal) include prohibitin (spot 1044), cathepsin D (spot 1044, 1045), and hexosaminase B (spot 1302). All 3 proteins become more prominent driving factors when nonmenopausal women are excluded, as indicated by the higher p(corr) values on the y-axis. Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Western blot images for validation of differentially expressed proteins (A, cathepsin B; B, lamin A/C). Experimental molecular weights (MW) obtained by using 2D-DIGE for cathepsin were 32 kDa for spot 1026 and 25 kDa for spots 1098 and 1113. Molecular weights obtained for lamin A/C include 68 kDa for spots 392, 395, and 397 and 64 kDa for spots 1313, 1587, 1600, and 1619. The dual molecular weights observed in the Western blots for each of the proteins (arrowheads) correspond with those measured by using 2D-DIGE for both proteins. A single band was detected for actin (45 kDa), which was used for normalization of the band volumes. Journal of Allergy and Clinical Immunology 2013 131, 743-751.e9DOI: (10.1016/j.jaci.2012.09.024) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions