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James N Baraniuk,1 Begona Casado,1 Lewis K Pannell,2 Peter B McGarvey,3 Piera Boschetto,4 Maurizio Luisetti,5,† Paolo Iadarola6 1Division of Rheumatology, Immunology and Allergy, Georgetown University, Washington, DC, 2Proteomics and Mass Spectrometry Laboratory, Mitchell Cancer Center, University of South Alabama, Mobile, AL, 3Innovation Center for Biomedical Informatics, Georgetown University, Washington, DC, USA; 4Department of Medical Sciences, University of Ferrara, Ferrara, 5SC Pneumologia, Dipartimento Medicina Molecolare, Fondazione IRCCS Policlinico San Matteo, 6Lazzaro Spallanzani Department of Biology and Biotechnology, University of Pavia, Pavia, Italy †Maurizio Luisetti passed away on October 20, 2014 Rationale: Subtypes of cigarette smoke-induced disease affect different lung structures and may have distinct pathophysiological mechanisms. Objective: To determine if proteomic classification of the cellular and vascular origins of sputum proteins can characterize these mechanisms and phenotypes. Subjects and methods: Individual sputum specimens from lifelong nonsmokers (n=7) and smokers with normal lung function (n=13), mucous hypersecretion with normal lung function (n=11), obstructed airflow without emphysema (n=15), and obstruction plus emphysema (n=10) were assessed with mass spectrometry. Data reduction, logarithmic transformation of spectral counts, and Cytoscape network-interaction analysis were performed. The original 203 proteins were reduced to the most informative 50. Sources were secretory dimeric IgA, submucosal gland serous and mucous cells, goblet and other epithelial cells, and vascular permeability. Results: Epithelial proteins discriminated nonsmokers from smokers. Mucin 5AC was elevated in healthy smokers and chronic bronchitis, suggesting a continuum with the severity of hypersecretion determined by mechanisms of goblet-cell hyperplasia. Obstructed airflow was correlated with glandular proteins and lower levels of Ig joining chain compared to other groups. Emphysema subjects’ sputum was unique, with high plasma proteins and components of neutrophil extracellular traps, such as histones and defensins. In contrast, defensins were correlated with epithelial proteins in all other groups. Protein-network interactions were unique to each group. Conclusion: The proteomes were interpreted as complex “biosignatures” that suggest distinct pathophysiological mechanisms for mucin 5AC hypersecretion, airflow obstruction, and inflammatory emphysema phenotypes. Proteomic phenotyping may improve genotyping studies by selecting more homogeneous study groups. Each phenotype may require its own mechanistically based diagnostic, risk-assessment, drug- and other treatment algorithms. Keywords: cigarette smokers, chronic bronchitis, emphysema, proteomics, mucous hypersecretion, mucin 5AC, neutrophil extracellular nets