Proteome profiling reveals novel biomarkers to identify complicated parapneumonic effusions

www.nature.com/scientificreports OPEN Received: 30 December 2016 Accepted: 2 June 2017 Published: xx xx xxxx Proteome profiling reveals novel biomarkers to identify complicated parapneumonic effusions Kuo-An Wu1,2, Chih-Ching Wu3,4,5, Chi-De Chen5, Chi-Ming Chu6, Li-Jane Shih7,8, Yu-Ching Liu3, Chih-Liang Wang9, Hsi-Hsien Lin10,11 & Chia-Yu Yang5,10,12 Patients with pneumonia and parapneumonic effusion (PPE) have elevated mortality and a poor prognosis. The aim of this study was to discover novel biomarkers to help distinguish between uncomplicated PPE (UPPE) and complicated PPE (CPPE). Using an iTRAQ-based quantitative proteomics, we identified 766 proteins in pleural effusions from PPE patients. In total, 45 of these proteins were quantified as upregulated proteins in CPPE. Four novel upregulated candidates (BPI, NGAL, AZU1, and calprotectin) were selected and further verified using enzyme-linked immunosorbent assays (ELISAs) on 220 patients with pleural effusions due to different causes. The pleural fluid levels of BPI, NGAL, AZU1, and calprotectin were significantly elevated in patients with CPPE. Among these four biomarkers, BPI had the best diagnostic value for CPPE, with an AUC value of 0.966, a sensitivity of 97%, and a specificity of 91.4%. A logistic regression analysis demonstrated a strong association between BPI levels > 10 ng/ml and CPPE (odds ratio = 341.3). Furthermore, the combination of pleural fluid BPI levels with LDH levels improved the sensitivity and specificity to 100% and 91.4%, respectively. Thus, our findings provided a comprehensive effusion proteome data set for PPE biomarker discovery and revealed novel biomarkers for the diagnosis of CPPE. A parapneumonic effusion (PPE) is an accumulation of exudative pleural fluid that occurs in association with an ipsilateral pulmonary infection. PPEs are present in 20% to 40% of hospitalized patients with pneumonia1. Based on fluid characteristics and pathogenesis, PPEs are classified into the following three groups: uncomplicated PPE (UPPE), complicated PPE (CPPE), and thoracic empyema2. The occurrence of CPPE or empyema greatly increases the risk of morbidity and mortality compared to that of UPPE1. Generally, UPPE can be cured with antibiotic treatment alone. When CPPE and empyema are present, pleural space drainage is mandatory2. For conditions such as patients with loculated pleural collection or poor clinical progress during treatment with antibiotics alone, drainage treatment will also be required3. Thus, an accurate diagnosis in the early stages of PPE is important for clinicians. The classical criteria for clinical diagnosis of PPE is based on the biochemical parameters of the pleural effusions, including levels of lactate dehydrogenase (LDH), glucose, and pH1. To improve PPE diagnosis, numerous biomarkers, such as inflammatory cytokines (tumour necrosis factor-alpha/TNF-α, interleukin-8/IL-8, and IL-1β), enzymes (neutrophil elastase, myeloperoxidase/MPO, and metalloproteinases/MMPs), C-reactive protein 1 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan. Department of Internal Medicine, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan. 3Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 4 Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 5 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan. 6Division of Biomedical Statistics and Informatics, School of Public Health, National Defense Medical Center, Taipei, Taiwan. 7Department of Medical Laboratory, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan. 8Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan. 9Division of Pulmonary Oncology and Interventional Bronchoscopy, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 10Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 11Chang Gung Immunology Consortium and Department of Anatomic Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 12Division of Colon and Rectal Surgery, Department of Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan. Kuo-An Wu and Chih-Ching Wu contributed equally to this work. Correspondence and requests for materials should be addressed to H.-H.L. (email: ) or C.-Y.Y. (email: ) 2 Scientific Reports | 7: 4026 | DOI:10.1038/s41598-017-04189-4 1 www.nature.com/scientificreports/ Figure 1. Schematic diagram of the experimental design for identifying novel biomarkers in parapneumonic effusions. Pleural effusions were collected from patients with different causes, including transudates, other exudates, malignant, uncomplicated parapneumonic effusion (UPPE) and complicated parapneumonic effusion (CPPE). Four samples from each type of UPPE or CPPE were pooled, processed, and labeled with iTRAQ reagents, following by 2D LC-MS/MS analysis. Peptide and protein were identified and quantified using MASCOT software. Selected proteins were uploaded into the DAVID database and MetaCore software to evaluate possible associations with known pathways and biological processes. The potential newer biomarkers were validated in an extended patients group by ELISA assays. Statistical analysis was performed to determine the efficacy of biomarkers in discriminating between UPPE and CPPE patients. (CRP), and soluble triggering receptor expressed on myeloid cells (sTREM-1), have been evaluated4–9. In these reports, the candidates were selected by a literature search and confirmed by immunoassays10. Currently, high-throughput proteomics technology provides more comprehensive proteome profiling of body fluids, which facilitates biomarker discovery11–13. Thus, the characterization of proteomic changes associated with PPE progression helps to elucidate disease mechanisms and identify useful biomarkers and therapeutic targets. In this study, we aimed to investigate useful biomarkers for the differential diagnosis of UPPE and CPPE, with the goal of identifying specific proteins and pathways important for the molecular mechanisms of PPE progression. Using the comprehensive proteomics approach, we generated the PPE proteome data set for biomarker research and verified the levels of four novel proteins (BPI, NGAL, AZU1, and calprotectin) in PPE. Collectively, we identified novel biomarkers for the diagnosis of CPPE. Results Proteome profiling of UPPE and CPPE by iTRAQ-based mass spectrometry. To identify novel bio- markers for PPE diagnosis, the proteomes of pleural effusions from patients with UPPE and CPPE (Supplementary Table 1) were analyzed using iTRAQ-based mass spectrometry (Fig. 1). After depletion of six abundant proteins (albumin, immunoglobulin G, immunoglobulin A, transferrin, α1-antitrypsin, and haptoglobin) using affinity column, the pleural effusion (...truncated)