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Quantitative proteomics of bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis Matthew W Foster J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr501149m • Publication Date (Web): 26 Dec 2014 Downloaded from http://pubs.acs.org on January 12, 2015
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Journal of Proteome Research
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Quantitative proteomics of bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis
Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:
Journal of Proteome Research pr-2014-01149m.R1 Article 22-Dec-2014 Foster, Matthew; Duke University Medical Center, Morrison, Lake; Duke University Medical Center, Todd, Jamie; Duke University Medical Center, Snyder, Laurie; Duke University Medical Center, Thompson, J.; Duke University, School of Medicine Soderblom, Erik; Duke University, School of Medicine Plonk, Kelly; Duke University Medical Center, Weinhold, Kent; Duke University Medical Center, Townsend, Robert; Bristol Myers-Squibb, EXPLORATORY CLINical & TRANSLATIONAL RESEARCH Minnich, Anne; Bristol Myers-Squibb, EXPLORATORY CLINical & TRANSLATIONAL RESEARCH Moseley, M.; Duke University,
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Quantitative proteomics of bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis Matthew W. Foster†,§,*, Lake D. Morrison†, Jamie L. Todd†, Laurie D. Snyder†, J. Will Thompson§, Erik J. Soderblom §, Kelly Plonk¶, Kent J. Weinhold¶, Robert Townsend║, Anne Minnich║ and M. Arthur Moseley§
†
Pulmonary, Allergy and Critical Care Medicine, §Duke Proteomics and Metabolomics Core Facility,
Department of Surgery, Duke University Medical Center, Durham NC and ║Exploratory Clinical and
¶
Translational Research, Bristol-Myers Squibb Co., Princeton NJ
*
To whom correspondence should be addressed:
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ABSTRACT 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.
KEYWORDS airway lining fluid, immunodepletion, targeted proteomics, label-free, PEDF, CRAC1
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INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating, and ultimately fatal scarring lung disease of unknown etiology.1 IPF belongs to a larger group of diffuse parenchymal lung diseases, several of which involve extracellular matrix accumulation and fibrous expansion within the pulmonary interstitium, between the alveolar epithelial cells and the vascular endothelium.2, 3 Common symptoms include shortness of breath, cough, a progressive restrictive ventilatory defect, and eventual development of hypoxemic respiratory failure, with or without secondary pulmonary hypertension and heart failure.4 IPF affects approximately five million people worldwide. The three-year survival rate of the disease is approximately 50%, and historically, lung transplant has been the only viable treatment option.5 Recently, two drugs, pirfenidone and nintedanib, have been approved by the FDA. These drugs have been shown to slow disease progression but not diseaserelated mortality.6,
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Given the impact of IPF on patients and the complexity of its pathogenesis, the
identification of molecular signatures of disease and disease progression should be invaluable to both the identification of potential drug targets and the quantification of early response to therapy in future clinical trials.8, 9 For the past ten or more years, the transcriptomic analyses of lung tissue,10-17 fibroblasts,18, 19 epithelium20 and even peripheral blood cells21 has been the overwhelming focus of molecular phenotyping and biomarker discovery in IPF. In turn, serum and plasma have been the matrices of choice for validation of putative predictive and prognostic biomarkers identified from these ‘omic analyses. These approaches can be rationalized by the fact that IPF is characterized by fibroblast proliferation and extracellular matrix deposition, and thus, lung tissue should be a sensitive milieu for identification of these and related pathological processes. Additionally, peripheral blood is among the easiest fluids to obtain and is a preferred sample type for molecular diagnostics. Nonetheless, changes in tissue gene expression may not be necessarily be reflected at the protein level, and it can be difficult to predict the gene(s) that may constitute peripheral markers of disease. Moreover, it is possible that many of the proteins dysregulated in IPF may not traverse the lung endothelial barrier or will be diluted out by more abundant constituents, and thus will be undetectable in plasma. Since type 2 alveolar 3 ACS Paragon Plus Environment
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epithelial cells are known to be abnormal in IPF and have been implicated in disease pathogenesis, the alveolar lining fluid, as sampled by bronchoalveolar lavage (BAL), may represent an equally or more sensitive compartment for protein biomarker discovery and diagnostic tests. In human IPF, to date, the proteomics analysis of BAL fluid (BALF) has been limited to two-dimensional gel electrophoresis (2D-GE) and MS identification of differentially-expressed spots.22-24 Such approaches are hindered by the presence of high-abundance plasma proteins and overall large dynamic range of protein concentrations in BALF, and they do not quantify protein expression changes for all identifiable proteins. Alternatively, shotgun proteomic approaches, which have not before been applied to the study of IPF, can simultaneously quantify hundreds of proteins in airway lining fluid and are readily coupled with immunodepletion of high-abundance plasma proteins to increase depth of proteome coverage. We recently developed a gel-free, label-free quantitative proteomic workflow for analysis of human BALF.25 Here, we applied this methodology to IPF, in order to better understand how global quantitation of the BALF proteome might be useful in identifying new therapeutic targets or disease markers. In the context of this analysis, we also sought to determine: 1) how sample handling might impact the quality of BALF samples for subsequent proteomic analysis; 2) whether on-line two-dimensional liquid chromatography separation of protein digests and ion mobility-assisted data independent acquisition could significantly improve depth of proteome coverage; and 3) whether targeted proteomics could have utility for secondary discovery and validation in BALF.
METHODS Subject inclusion criteria. Studies were performed according to Duke University Institutional Review Board protocol. Inclusion criteria for normal controls were: aged 40-80 years, BMI 18-35, force vital capacity (FVC) > 80%; forced expiratory volume in 1 second (FEV1) > 80%; FEV1/FVC ratio > 70%; diffusing capacity of the lung for carbon monoxide (DLCO) > 80%; and chest x-ray showing no pulmonary, pleural, or mediastinal abnormalities. Inclusion criteria for patients with IPF included: aged 50-80 years; BMI 18-35; diagnosis of IPF, by surgical lung biopsy (SLB) or high resolution CT (HRCT), consistent with current American Thoracic 4 ACS Paragon Plus Environment
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Society/European Respiratory Society guidelines26 and no features supporting an alternative diagnosis; FVC 5080%; DLCO 30-80%. Subjects also had no evidence of improvement in IPF severity over the preceding year. Bronchoalveolar lavage. BAL was performed sequentially on right lower lobe (RLL) and right middle lobe (RML). BAL was first performed on any subsegment of the RLL (except the posterior basal segment). Lavage was performed with 5 x 20 ml aliquots of room temperature saline. Return from the first aliquot was discarded, and the return from aliquots 2-5 were combined. Next, BAL was performed on the RML until a combined volume (RLL + RLL) of 100 ml was reached, or until a total maximum volume of 300 ml of saline was instilled. The BAL fluid was filtered through sterile gauze followed by centrifugation at 300 xg for 10 min to recover airway cells. A Micro-BCA assay (Pierce) was performed on 50 µl of BALF to determine total protein recovered. Cell-free supernatants were frozen at -80 ºC until all samples had been collected. Protein concentration and immunodepletion. BAL fluid was thawed and concentrated from 10 ml to a volume of 200-400 µl with Millipore Amicon Ultra-4 (10 kDa cut-off) centrifugal filters. Filters were prewashed with 50 mM ammonium bicarbonate (AmBic) prior to use. Bradford assays (Coomassie Plus Protein Assay Reagent; Pierce) were performed after concentration and samples were normalized to 760 µl with AmBic followed by addition of 190 µl of HC Sample Dilution buffer (Agilent). Following filtration with a 0.2 µm spin column, samples were immunodepleted using a MARS-14 LC column (Agilent) and Agilent 1000 HPLC. Unbound fractions were concentrated to ~50 µl followed by two buffer exchanges with 1 ml AmBic. Finally, a Bradford assay was performed on the samples post-depletion. To visualize immunodepeletion, 5 µg of samples were diluted to 30 µl with AmBic containing 10 mM DTT, followed by addition of 10 µl of 4x NuPage LDS Sample Buffer (Invitrogen). Protein was separated using NuPage 4-12% Bis-Tris Gels (Invitrogen) with MES SDS Running Buffer and stained with Colloidal Blue Stain (Invitrogen). 2D-LC-MS/MS analysis. 15 µg of each sample was reduced and denatured by heating with 10 mM DTT and 0.2 % RapiGest-SF (Waters) at 80 °C for 10 min. Protein was alkylated with 25 mM iodoacetamide for 30 min in the dark. Finally, the protein was digested with 1:50 (w/w) Sequencing Grade Modified Trypsin (Promega) at 37 °C overnight in a Eppendorf Thermomixer. Digested samples were incubated with 1% trifluoroacetic acid 5 ACS Paragon Plus Environment
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(TFA), 2 % acetonitrile (MeCN) at 60 °C for 2 h to degrade the RapiGest. Samples were lyophilized to dryness in a Speed-Vac. Samples were resuspended at 1 µg/µl in 200 mM Ammonium Formate (pH 10) containing 25 fmol/µl trypsinized yeast alcohol dehydrogenase 1 (MassPrep, Waters) as surrogate standard. A QC Pool, for use for column conditioning and technical reproducibility assessment, was made by mixing 5 µl from each of the twelve samples. Quantitative two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) utilized a high-low pH reversed phase/reversed phase configuration on a nanoAcquity UPLC/UPLC system (Waters Corp) coupled to a Synapt G2 HDMS mass spectrometer (Waters Corp) with nanoelectrospray ionization as previously described.27 Briefly, 3 µg of each sample was injected at 2 µl/min onto a 5 µm XBridge BEH130 C18 300 um × 50 mm column (Waters) at 97/3 v/v water/MeCN in 20 mM ammonium formate (pH 10). Peptides were eluted at 2 µl/min in five steps of 10.8%, 14.0%, 16.7%, 20.4% and 50.0% MeCN, and each of these fractions were separately diluted 10-fold online with 99.8/0.1/0.1 v/v/v water/MeCN/formic acid and trapped on a 5 µm Symmetry C18 180 µm × 20 mm column (Waters), followed by 2nd dimension separations were performed on a 1.7 µm Acquity BEH130 C18 75 µm × 150 mm column (Waters) using a linear gradient of 7 to 35% MeCN with 0.1% formic acid over 37 min, at a flow rate of 0.5 µl/min and column temperature of 35 °C. Data collection was performed in data-dependent acquisition (DDA) and ion-mobility assisted dataindependent acquisition (HDMSE) modes. DDA mode used 0.6 s MS scan followed by MS/MS acquisition on the top 3 ions with charge greater than 1, and HDMSE mode used a 0.6 s alternating cycle time between low (6V) and high (27-50V) collision energy (CE) . Data Analysis. Data was imported into Rosetta Elucidator v3.3 (Rosetta Biosoftware, Inc), and aligned based on the accurate mass and retention time of detected ions (“features”) using PeakTeller algorithm in Elucidator. Relative peptide abundance was calculated based on area-under-the-curve (AUC) of aligned features across all runs. DDA and HDMSE data were searched with Mascot Distiller v2.2 and ProteinLynx Global Server v2.5.2, respectively, against a Swiss-Prot database with Homo sapiens taxonomy which contained an equal number of reversed-sequence “decoy” entries for false positive rate determination (downloaded on 12/07/10; 40,530 6 ACS Paragon Plus Environment
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unique entries including 50% reverse sequences). Database searches used fixed modification on Cys (carbamidomethyl) and variable modifications on Met (oxidation) and Asn/Gln (deamidation). Variable modification on Pro (hydroxyl) was also included in the Mascot search. Searches allowed for up to 2 missed cleavages with trypsin specificity. After individual peptide scoring using PeptideProphet algorithm (Elucidator), the data was annotated at a
