Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs)
Small urinary extracellular vesicles (suEVs) are 50–200 nm membrane-delimited vesicles secreted mainly by urothelial cells. suEVs have become a promising non-invasive source of biomarkers for urinary diseases. However, suEV proteomic studies are limited due to the low concentration of EVs in urine s...
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| Language: | English |
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Elsevier
2025-06-01
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| Series: | Extracellular Vesicle |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2773041725000125 |
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| author | Yilan Hu Jifeng Wang Xiaoqing Qing Tanxi Cai Lili Niu Xiang Ding Zhensheng Xie Mengmeng Zhang Xiaojing Guo Xiulan Chen Fuquan Yang |
| author_facet | Yilan Hu Jifeng Wang Xiaoqing Qing Tanxi Cai Lili Niu Xiang Ding Zhensheng Xie Mengmeng Zhang Xiaojing Guo Xiulan Chen Fuquan Yang |
| author_sort | Yilan Hu |
| collection | DOAJ |
| description | Small urinary extracellular vesicles (suEVs) are 50–200 nm membrane-delimited vesicles secreted mainly by urothelial cells. suEVs have become a promising non-invasive source of biomarkers for urinary diseases. However, suEV proteomic studies are limited due to the low concentration of EVs in urine samples and poor proteomic coverage caused by high abundant uromodulin. In this study, we compared four methods for suEV isolation, including ultracentrifugation (UC), ultracentrifugation with DTT treatment (DTT + UC), filtration and ultracentrifugation (F + UC), and filtration and ultrafiltration (F + UF). We evaluated their recovery, EV purity, and proteomic coverage using multiple techniques. The combination of filtration and ultracentrifugation (F + UC) showed the best performance with efficient removal of uromodulin fibers and successful in-depth proteome identification. Furthermore, we performed a deep-going proteomic analysis and characterized suEV subsets purified by the four methods. Lastly, we developed a statistical approach to evaluate universal suEV proteins, independent of the isolation techniques used, by calculating the correlation between protein abundance and sample purity. This study provided an integrated workflow for the isolation and proteomic analysis of suEVs, which could facilitate clinical biomarker discovery and diagnosis in urology disease. |
| format | Article |
| id | doaj-art-90bf079bea224e799666855146ea120e |
| institution | Kabale University |
| issn | 2773-0417 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Extracellular Vesicle |
| spelling | doaj-art-90bf079bea224e799666855146ea120e2025-08-20T03:46:58ZengElsevierExtracellular Vesicle2773-04172025-06-01510007610.1016/j.vesic.2025.100076Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs)Yilan Hu0Jifeng Wang1Xiaoqing Qing2Tanxi Cai3Lili Niu4Xiang Ding5Zhensheng Xie6Mengmeng Zhang7Xiaojing Guo8Xiulan Chen9Fuquan Yang10Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, ChinaLaboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China; Corresponding author. Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China; Corresponding author. Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.Small urinary extracellular vesicles (suEVs) are 50–200 nm membrane-delimited vesicles secreted mainly by urothelial cells. suEVs have become a promising non-invasive source of biomarkers for urinary diseases. However, suEV proteomic studies are limited due to the low concentration of EVs in urine samples and poor proteomic coverage caused by high abundant uromodulin. In this study, we compared four methods for suEV isolation, including ultracentrifugation (UC), ultracentrifugation with DTT treatment (DTT + UC), filtration and ultracentrifugation (F + UC), and filtration and ultrafiltration (F + UF). We evaluated their recovery, EV purity, and proteomic coverage using multiple techniques. The combination of filtration and ultracentrifugation (F + UC) showed the best performance with efficient removal of uromodulin fibers and successful in-depth proteome identification. Furthermore, we performed a deep-going proteomic analysis and characterized suEV subsets purified by the four methods. Lastly, we developed a statistical approach to evaluate universal suEV proteins, independent of the isolation techniques used, by calculating the correlation between protein abundance and sample purity. This study provided an integrated workflow for the isolation and proteomic analysis of suEVs, which could facilitate clinical biomarker discovery and diagnosis in urology disease.http://www.sciencedirect.com/science/article/pii/S2773041725000125Small urinary extracellular vesiclesProteomicsUromodulinUltracentrifugationMembrane filtration |
| spellingShingle | Yilan Hu Jifeng Wang Xiaoqing Qing Tanxi Cai Lili Niu Xiang Ding Zhensheng Xie Mengmeng Zhang Xiaojing Guo Xiulan Chen Fuquan Yang Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) Extracellular Vesicle Small urinary extracellular vesicles Proteomics Uromodulin Ultracentrifugation Membrane filtration |
| title | Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) |
| title_full | Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) |
| title_fullStr | Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) |
| title_full_unstemmed | Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) |
| title_short | Optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles (suEVs) |
| title_sort | optimization and assessment of an integrated workflow for the isolation and proteomic analysis of small urinary extracellular vesicles suevs |
| topic | Small urinary extracellular vesicles Proteomics Uromodulin Ultracentrifugation Membrane filtration |
| url | http://www.sciencedirect.com/science/article/pii/S2773041725000125 |
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