Research Article: iTRAQ-Based Proteomics Reveals Novel Biomarkers for Idiopathic Pulmonary Fibrosis

Date Published: January 25, 2017

Publisher: Public Library of Science

Author(s): Rui Niu, Ying Liu, Ying Zhang, Yuan Zhang, Hui Wang, Yongbin Wang, Wei Wang, Xiaohui Li, Oliver Eickelberg.


Idiopathic pulmonary fibrosis (IPF) is a gradual lung disease with a survival of less than 5 years post-diagnosis for most patients. Poor molecular description of IPF has led to unsatisfactory interpretation of the pathogenesis of this disease, resulting in the lack of successful treatments. The objective of this study was to discover novel noninvasive biomarkers for the diagnosis of IPF. We employed a coupled isobaric tag for relative and absolute quantitation (iTRAQ)-liquid chromatography–tandem mass spectrometry (LC–MS/MS) approach to examine protein expression in patients with IPF. A total of 97 differentially expressed proteins (38 upregulated proteins and 59 downregulated proteins) were identified in the serum of IPF patients. Using String software, a regulatory network containing 87 nodes and 244 edges was built, and the functional enrichment showed that differentially expressed proteins were predominantly involved in protein activation cascade, regulation of response to wounding and extracellular components. A set of three most significantly upregulated proteins (HBB, CRP and SERPINA1) and four most significantly downregulated proteins (APOA2, AHSG, KNG1 and AMBP) were selected for validation in an independent cohort of IPF and other lung diseases using ELISA test. The results confirmed the iTRAQ profiling results and AHSG, AMBP, CRP and KNG1 were found as specific IPF biomarkers. ROC analysis indicated the diagnosis potential of the validated biomarkers. The findings of this study will contribute in understanding the pathogenesis of IPF and facilitate the development of therapeutic targets.

Partial Text

Idiopathic pulmonary fibrosis (IPF), a devastating form of chronic interstitial lung diseases with an unfavorable outcome leading to respiratory failure, is a chronic fibrosing interstitial pneumonia encountered by clinicians in the intensive care units [1, 2]. Since its description, significant research has been achieved to advance our knowledge about its pathogenesis, epidemiology and treatment options. Nevertheless, there are no hitherto established drug therapies because suitable symptoms for initiating therapy, best candidates for treatment, and conceivable role for combinatory therapies are still controversial [3, 4]. The mortality from IPF is nearly 40% with a median survival of 2 to 3 year [5, 6]. This high mortality is partly due to incomplete understanding of the molecular mechanisms that are specifically lacking in the IPF patients who do not recover from their illness. To address this gap in knowledge, it is essential to characterize the protein expression in order to identify the proteins and the biological processes that are different in the IPF patients vs. healthy subjects in order to facilitate earlier and correct diagnosis of IPF for establishing effective treatment and prolonging survival.

Molecular biomarkers are highly desired in idiopathic pulmonary fibrosis (IPF) where they hold the potential to elucidate underlying disease mechanisms, accelerated drug development, and advance clinical management [8]. At present, there are no molecular biomarkers made available in well-known clinical application for IPF, and the research for discovery of potential markers remains in its early stages. Therefore, screening differentially expressed serum proteins in patients with IPF could provide an effective way to identify diagnostic biomarkers. In previous studies, a set of IPF biomarkers have been developed by applying proteomics, transcriptomics and genomics approaches to the analysis of lung tissue, bronchoalveolar lavage fluid (BALF) or serum for patients with IPF. For instance, high levels of matrix metalloproteinase-8 (MMP-8), pepsin, BALF YKL-40, S100A9 and MRP14 have been found elevated in the BALFs of IPF patients [15, 28–31]. Similarly, elevated serum levels of vascular endothelial growth factor (VEGF), MMP3, CXCL13, heat shock protein 47 (HSP47) and several other proteins were equally reported in IPF [7, 13, 15, 30, 32–42]. In the present study, iTRAQ labeling coupled with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in serum of IPF patients. A total of 97 differentially expressed proteins were identified and eight different proteins including HBB, CRP, SERPINA1, APOA2, AHSG, KNG1 and AMBP were validated by ELISA. Finally, we evaluated the diagnostic accuracy of validated differentially expressed proteins and found that AHSG, CRP, SERPINA1, AMBP and KNG1 may be potential specific diagnostic biomarkers for IPF. In the present work, CRP was found as the most upregulated protein in the serum of IPF patients but its application as IPF biomarker still constitutes a problematic since it has been associated to various diseases such as chronic obstructive lung disease and lung cancer, obliterative bronchiolitis and systemic lupus erythematosus [43–47]. The functional role of APOA2, the most downregulated differentially expressed proteins identified in the present study, has not been reported in IPF in the past. However, low concentrations APOA1 were found in bronchoalveolar lavage fluids from subjects with IPF [48]. This suggested the involvement of APOA2 in the pathophysiology of IPF. In this study, we found hemoglobin (HBB) as a biomarker for IPF. This should be taken with precaution because hemoglobin is generally regarded as a marker for lysis of erythrocytes during sample draw [49]. Intriguingly, most of previously reported IPF biomarkers [7–16, 28–42, 50–70] were not identified in our study. Therefore, the present findings are promising for the discovery and development of novel biomarkers for IPF.

In conclusion, the use of iTRAQ labeling of serum samples from IPF patients in combination with LC-MS/MS allowed the discovery of numerous novel, differentially expressed proteins between IPF patients and normal controls. A preliminary analysis of the specificity of some of these proteins exhibited good discriminatory power against other lung diseases. The new potential biomarkers warrant further in-depth experimentation to be validated as IPF biomarkers.