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Quantitative Proteomics of Bronchoalveolar Lavage Fluid in Idiopathic Pulmonary Fibrosis

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Pulmonary, Allergy and Critical Care Medicine, Duke Proteomics and Metabolomics Shared Resource, §Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, United States, and
Exploratory Clinical and Translational Research, Bristol-Myers Squibb Co., Princeton New Jersey 08543, United States
Cite this: J. Proteome Res. 2015, 14, 2, 1238–1249
Publication Date (Web):December 26, 2014
https://doi.org/10.1021/pr501149m
Copyright © 2014 American Chemical Society

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    Abstract

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    The proteomic analysis of bronchoalveolar lavage fluid (BALF) can give insight into pulmonary disease pathology and response to therapy. Here, we describe the first gel-free quantitative analysis of BALF in idiopathic pulmonary fibrosis (IPF), a chronic and fatal scarring lung disease. We utilized two-dimensional reversed-phase liquid chromatography and ion-mobility-assisted data-independent acquisition (HDMSE) for quantitation of >1000 proteins in immunodepleted BALF from the right middle and lower lobes of normal controls and patients with IPF. Among the analytes that were increased in IPF were well-described mediators of pulmonary fibrosis (osteopontin, MMP7, CXCL7, CCL18), eosinophil- and neutrophil-derived proteins, and proteins associated with fibroblast foci. For additional discovery and targeted validation, BALF was also screened by multiple reaction monitoring (MRM), using the JPT Cytokine SpikeMix library of >400 stable isotope-labeled peptides. A refined MRM assay confirmed the robust expression of osteopontin, and demonstrated, for the first time, upregulation of the pro-fibrotic cytokine, CCL24, in BALF in IPF. These results show the utility of BALF proteomics for the molecular profiling of fibrotic lung diseases and the targeted quantitation of soluble markers of IPF. More generally, this study addresses critical quality control measures that should be widely applicable to BALF profiling in pulmonary disease.

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    Supplemental Excel file: Table S1. Summary of patient demographics and pulmonary function tests; Table S2. Protein concentrations and immunodepletion metrics. Table S3. Peptide expression data. Table S4. Protein expression data. Table S5. Protein fold changes in RLL-IPF-2 versus other IPF samples. Table S6. Protein fold changes in RML-Control-2 versus other control samples. Table S7. MRM data extracted from Skyline (data are expressed as light/heavy ratios). Supplemental PDF file: Supplemental Methods. Figure S1. Freeze–thaw reproducibility study. (A) Schematic of freeze–thaw reproducibility study (see Supplemental Methods). (B) SDS-PAGE analysis following the freeze–thaw and immunodepletion study. Figure S2. Principal component analysis. Figure S3. Hierarchical clustering of differentially expressed proteins. An unpaired t test was performed using expression data from control and IPF samples following exclusion of an outlier lobe (IPF-RLL-2), and the 192 proteins with p < 0.05 were visualized by 2D hierarchical clustering. Figure S4. Magnification of cluster analysis from Figure S3. This material is available free of charge via the Internet at http://pubs.acs.org. Electronic Data: Raw data from the 2D-LC-MS/MS analysis is available for download at Chorus (https://chorusproject.org) under the project name “IPF Proteomics”. Skyline files containing MRM methods and results have been made public as part of the Panorama Targeted Proteomics data repository (panoramaweb.org). (79) Skyline data from the plasma and BALF screening can be viewed at: http://goo.gl/t3NGG0 or downloaded at http://goo.gl/DoS8ue. Skyline data from the analysis of individual BALF samples can be viewed at http://goo.gl/mxKF15 or downloaded at http://goo.gl/s77BTG.

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