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Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation

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Abstract

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

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Funding

The work was primarily supported by research funding provided by the National Natural Science Foundation of China (31741036, 31700702, 21904098), the National Natural Science Foundation of China (Nos. U1703251 and U1810113), the Key Research and Development Plan of Shaanxi Province (No. 2017ZDCXL-GY-10-01-02), the Key Laboratory of Coal Resources Exploration and Comprehensive Utilization, Ministry of Land and Resources Open Research Topic (No. KF2016-4) and Shaanxi Provincial Innovation Capability Support Program (No. 2019TD-021), the Zhejiang Provincial and Ministry of Health Research Fund for Medical Sciences (WKJ-ZJ-1910), the Wenzhou Medical University (89218012, 89219012).

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Author notes

  1. Meng Li and Doudou Lou contributed equally to this work.

Authors and Affiliations

  1. College of Chemistry and Chemical Engineering, Xi’an University of Science and Technology, Xi’an, 710054, Shaanxi, China

    Meng Li & Yating Zhang

  2. School of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China

    Meng Li, Doudou Lou, Qingfu Zhu & Fei Liu

  3. Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, 325001, Zhejiang, China

    Meng Li, Yong Wang & Fei Liu

  4. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA

    Joyce Chen

  5. The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China

    Keqing Shi

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  1. Meng Li
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Li, M., Lou, D., Chen, J. et al. Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation. Anal Bioanal Chem 413, 365–375 (2021). https://doi.org/10.1007/s00216-020-03004-w

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