Analysis of Purity and Quality of Small Extracellular Vesicles Isolated from Blood Plasma by Proteomics and Raman Spectroscopy

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Vasiliy S. Chernyshev^1,2,a*, Natalia L. Starodubtseva^1,3, Elena N. Rimskaya^1,4, Anna E. Bugrova^1,5, Alexey S. Kononikhin^1,2, Denis N. Silachev^1,6, Alisa O. Tokareva¹, Ekaterina A. Evtushenko⁷, Alexander A. Yakovlev⁸, Alexander M. Yurin¹, Maria A. Kepsha¹, Elena A. Mezhevitinova¹, Eugene N. Nikolaev², Vladimir E. Frankevich¹, Niso M. Nazarova¹, Vera N. Prilepskaya¹, and Gennadiy T. Sukhikh^1,9

¹Federal State Budgetary Institution “National Medical Research Center for Obstetrics, Gynecology, and Perinatology Named after Academician V. I. Kulakov”, Ministry of Health of the Russian Federation, 117997 Moscow, Russia

²Skolkovo Institute of Science and Technology, 143026 Moscow, Russia

³Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia

⁴Lebedev Physical Institute, 119991 Moscow, Russia

⁵Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia

⁶A. N. Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁷Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁸Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia

⁹Department of Obstetrics, Gynecology, Perinatology and Reproductology, Institute of Professional Education, Federal State Autonomous Educational Institution of Higher Education I. M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received: November 18, 2025; Revised: February 13, 2026; Accepted: February 14, 2026

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Ultracentrifugation (UC) has long been considered the “gold standard” for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-intensive protocol, low yield considering initial biofluid volume and low throughput, development of new EV isolation approaches is still ongoing. Here we compare three methods for isolating the most studied EV subtype, small extracellular vesicles (sEVs), from human plasma: ultracentrifugation (UC), express asymmetric depth filtration (ExADFi), and anti-CD9 immunoaffinity capture (AS-CD9) with focus on their Raman and proteomic profiles. For all three methods, purity and quality of the sEV isolation were assessed based on the level of contamination of the sEV fraction with major plasma proteins such as albumin and apolipoproteins (APOA1, APOH, APOA4, APOC2, APOC1, and APOC4). UC showed the highest ratio of protein to nanoparticle concentration. AS-CD9 and ExADFi provided comparable to UC purity and levels of non-vesicular contaminants with AS-CD9 requiring minimal time and labor. ExADFi showed characteristics including purity of the sEV samples, yield, and isolation time that is between the UC and AS-CD9 methods. Raman spectroscopy provided more details about characteristics of the isolated sEVs and confirmed differences observed in the proteomic profiles. The findings demonstrate that the AS-CD9 and ExADFi methods could be appropriate substitutes of the classical UC-based isolation method and be chosen depending on the final requirements and use of the purified sEVs such as further functional and biomarker studies.

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KEY WORDS: small extracellular vesicles, isolation, blood plasma, proteomics, mass spectrometry, Raman spectroscopy

DOI: 10.1134/S0006297925603946

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