Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres

Abstract

There is an unmet need for novel biomarkers in the diagnosis of multifactorial COPD. We applied next-generation proteomics to serum extracellular vesicles (EVs) to discover novel COPD biomarkers.EVs from 10 patients with COPD and six healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics.Using non-targeted proteomics, we identified 406 proteins, 34 of which were significantly upregulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodelling. We also identified 63 upregulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl-peptidase 2, fibulin-1, and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD. Moreover, six proteins; fibulin-3, tripeptidyl-peptidase 2, UTP-glucose-1-phosphate uridylyl transferase, CD81, CD177, and oncoprotein-induced transcript 3, were correlated with emphysema. Upregulation of fibulin-3 was confirmed by immunoblotting of EVs and immunohistochemistry in lungs. Strikingly, fibulin-3 knockout mice spontaneously developed emphysema with age, as evidenced by alveolar enlargement and elastin destruction.We discovered potential pathogenic biomarkers for COPD using next-generation proteomics of EVs. This is a novel strategy for biomarker discovery and precision medicine.

Extracellular vesicles and chronic obstructive pulmonary disease (COPD): a systematic review

“…COPD) can alter the composition and packaging of proteins, mRNA and ncRNAs in EVs/exosomes Ex vivo Human In vitro RNA-seq analysis carried out on EVs from plasma samples of human subjects showed significant miRNAs up- or down-regulated in smokers vs. COPD and non-smokers vs. COPD pairwise comparisons Jung et al, 2020 [ 54 ] Surface proteome of plasma extracellular vesicles as biomarkers for pneumonia and acute exacerbation of chronic obstructive pulmonary disease Identify surface proteins of plasma small EVs (ssEVs) as biomarkers for diagnosis and differentiation of AECOPD to CAP (community acquired pneumonia) Ex vivo Human There was a significantly higher expression in plasma sEVs (CD45, CD28, CTLA4, TNF-R-II, and CD16) from patients with AECOPD when compared to CAP patients, allowing for discrimination between the two Koba et al . , 2021 [ 55 ] Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres Assess serum EVs to find novel biomarkers for personalised medicine in COPD using the latest proteomic strategies Ex vivo Human In vivo Mice This study identified novel biomarkers for COPD using next-generation proteomics of serum extracellular vesicles. Notably, the expression of fibulin-3 is correlated with lung function and emphysema Soni et al, 2021 [ 56 ] Intra-alveolar neutrophil-derived microvesicles are associated with disease severity in COPD Evaluate the profiles of intra-alveolar (within BALF) and circulating (within plasma) MVs in COPD patients, characterizing a variety of MV subtype populations Ex vivo Human This study identified a variety of MV subtype populations within the BALF and plasma of COPD patients with a spectrum of disease severity.…”

“…Shen et al [ 60 ] observed that expression levels of three exosomal miRNAs were negatively associated with FEV1. Koba et al [ 55 ] study observed that expression of fibulin-3 correlated with lung function and emphysema. Together these studies indicate that EVs can lead to the development of more accurate biomarkers to diagnose COPD and monitor disease progression.…”

Identification of CD14 and lipopolysaccharide-binding protein as novel biomarkers for sarcoidosis using proteomics of serum extracellular vesicles

“…3A ; Table 2 ; Supplementary Table 2 ). Targeted proteomics enables the efficient and specific verification of candidates without requiring antibodies and is, thus, a powerful tool for biomarker validation ( 14 , 15 , 20 ). Among the 25 biomarker candidates, the expression levels of LBP and the monocyte differentiation antigen CD14 were markedly elevated in patients with sarcoidosis ( Fig.…”

“…Some comparative studies have shown that the isolation of EVs by SEC retains the biophysical properties of EVs, and results in a higher yield ( 13 ). Tandem mass tag-based non-targeted proteomic analysis of serum EVs isolated by ultracentrifugation revealed that fibulin-3 in EVs may serve as a novel diagnostic biomarker for chronic obstructive pulmonary disease (COPD) and might be closely related to its pathophysiology ( 14 ). With this background, we isolated serum EVs using SEC, which can detect less abundant serum proteins, and applied label-free proteomic strategies to discover novel biomarkers for sarcoidosis.…”

Distinct Proteomic Profiling of Plasma Extracellular Vesicles from Moderate-to-Severe Atopic Dermatitis Patients

“… 13 , 14 Although multiple sample resources are available, peripheral blood has been regarded as an ideal source due to its reproducibility of measurement and feasibility of access. 15 However, 99% of serum proteins, including albumin, immunoglobulins, and complement factors, will mask some biomarker candidates when measured by conventional proteomics. To break down these barriers, we focused on proteomic profiling of EVs.…”