Koba Taro, Takeda Yoshito, Narumi Ryohei, Shiromizu Takashi, Nojima Yosui, Ito Mari, Kuroyama Muneyoshi, Futami Yu, Takimoto Takayuki, Matsuki Takanori, Edahiro Ryuya, Nojima Satoshi, Hayama Yoshitomo, Fukushima Kiyoharu, Hirata Haruhiko, Koyama Shohei, Iwahori Kota, Nagatomo Izumi, Suzuki Mayumi, Shirai Yuya, Murakami Teruaki, Nakanishi Kaori, Nakatani Takeshi, Suga Yasuhiko, Miyake Kotaro, Shiroyama Takayuki, Kida Hiroshi, Sasaki Takako, Ueda Koji, Mizuguchi Kenji, Adachi Jun, Tomonaga Takeshi, Kumanogoh Atsushi
Dept of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan.
ERJ Open Res. 2021 Mar 22;7(1). doi: 10.1183/23120541.00658-2020. eCollection 2021 Jan.
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, 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.
在多因素慢性阻塞性肺疾病（COPD）的诊断中，对新型生物标志物存在未满足的需求。我们将下一代蛋白质组学应用于血清细胞外囊泡（EVs），以发现新型COPD生物标志物。通过基于串联质量标签的非靶向蛋白质组学分析了10例COPD患者和6例健康对照者的EVs，并通过非靶向蛋白质组学分析了弹性蛋白酶处理的肺气肿小鼠模型的EVs。为了进行验证，通过靶向蛋白质组学对23例COPD患者和20例健康对照者的EVs进行了验证。使用非靶向蛋白质组学，我们鉴定出406种蛋白质，其中34种在COPD患者中显著上调。值得注意的是，COPD患者的EV蛋白质特征反映了炎症和重塑。通过分析从小鼠模型中分离的EVs，我们还从1956种蛋白质中鉴定出63种上调的候选物。结合人和小鼠的生物标志物候选物，我们通过靶向蛋白质组学、选择反应监测验证了45种蛋白质。值得注意的是，纤维连接蛋白-3、三肽基肽酶2、纤维连接蛋白-1和富含半胱氨酸结构域的可溶性清道夫受体蛋白在COPD患者中的水平显著更高。此外，六种蛋白质；纤维连接蛋白-3、三肽基肽酶2、尿苷三磷酸-葡萄糖-1-磷酸尿苷酰转移酶、CD81、CD177和癌蛋白诱导转录物3，与肺气肿相关。通过对EVs的免疫印迹和肺组织的免疫组织化学证实了纤维连接蛋白-3的上调。令人惊讶的是，敲除小鼠随着年龄的增长自发地发展为肺气肿，表现为肺泡扩大和弹性蛋白破坏。我们使用EVs的下一代蛋白质组学发现了COPD的潜在致病生物标志物。这是一种用于生物标志物发现和精准医学的新策略。
