Integrative Use of Mucus Brushes, Induced Sputum, and Broncho-alveolar Lavage: Towards a Holistic Profiling of Mucins and Regional Lung Biomarkers

Abstract Rationale: The lung epithelia's mucosal barrier plays a vital role in respiratory health, secreting thousands of proteins with diverse lung-physiological functions, including the protective gel-forming mucins MUC5AC and MUC5B. Notably, the lung surface encompasses at least three major regions —the proximal/large airways, the distal/small airways, and the alveolar region — each requiring specific mucus compositions. The premise of this investigation is to characterize the protein compositions across these regions to better understand their contributions to lung health and diseases, by comparing common lung secretion sampling methods. Methods: Utilizing bronchial brush mucus (BBM), induced sputum (IS) and broncho-alveolar lavage (BAL) samples from 41 non-smoker and 43 smoker UNC TCORS study participants, we analyzed the distribution profiles of secreted and other proteins by label-free mass spectrometry based quantitative proteomics. Total precursor intensities were normalized across all samples to allow for relative protein level comparisons across sample types. Results: The proteomic analysis identified ∼2000 proteins including secreted, cell surface, and intracellular proteins. Principal component analysis indicated that BBM, IS and BAL sample groups show unique protein compositions, with overlap between BAL and IS, and BBM exhibiting the most distinct clustering from the former two. BAL samples displayed the highest level of serum proteins, such as albumin, serotransferrin, alpha-1-antitrypsin, or antithrombin III. When using the distribution profile of the alveolar region-specific surfactant protein SFTPD, other proteins, e.g. C1QC, GALNT5 and APOH showed similar alveolar distributions. BBM contained the most cell surface related proteins including cilia proteins, e.g. IFTs and rootletin, likely due to epithelial surface contact during brush collection. In terms of the gel-forming mucins, MUC5B was most abundant in IS, whereas MUC5AC was markedly higher in BBM, resulting in a higher MUC5AC/MUC5B ratio in BBM. Among smokers, smoking markers such as ALDH3A1 and MUC5AC were increased across all three sampling methods. Conclusions: BBM, IS and BAL exhibit distinct qualitative and quantitative proteomes. The distributions of protein markers — including mucins, secretoglobins and surfactant proteins — suggest that BBM samples mainly represent the large airways, IS samples mostly small airways and BAL the alveolar region. Notably, in smokers, the ratio of MUC5AC/MUC5B increased significantly across all sampling methods, and most dramatically in BBM samples. Overall, our study provides a more holistic profiling of the lung mucus proteome, including regional and diagnostic markers. Our protein mapping may inform sampling approaches for future investigations, including airway remodeling and the “proximalization” of the distal airways in diseases.