Comparative evaluation of five extracellular vesicle isolation methods using proteomic profiling

Klara Mrazova; Tomas Henek; Zuzana Mihal Jurikova; Simona Koznarova; Michaela Vasinova Galiova; Jaromir Bacovsky; Lenka Hernychova; Roman Hrstka; Klara Mrazova; Tomas Henek; Zuzana Mihal Jurikova; Simona Koznarova; Michaela Vasinova Galiova; Jaromir Bacovsky; Lenka Hernychova; Roman Hrstka

doi:10.3934/molsci.2026006

AIMS Molecular Science

2026, Volume 13, Issue 1: 97-118. doi: 10.3934/molsci.2026006

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Research article

Comparative evaluation of five extracellular vesicle isolation methods using proteomic profiling

1.
Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53 Brno, Czech Republic
2.
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic
3.
Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic
4.
DELONG INSTRUMENTS a.s., Palackého třída 153b, 612 00 Brno, Czech Republic

Received: 20 December 2025 Revised: 09 February 2026 Accepted: 27 February 2026 Published: 18 March 2026

Extracellular vesicles (EVs) research has gained a significant amount of attention in recent years. EVs are a heterogeneous group of different vesicles that vary in their origin, size, and function. The very nature of EVs, from their biogenesis to the contents of their cargo, offers many options for exploration. Despite being studied for a few decades now, there has not been an established standardized approach to isolation. However, the future use of EVs in clinical practice is conditioned by the optimization and standardization of isolation methods. In this study, we systematically compared common EV isolation techniques such as ultracentrifugation, precipitation, tangential flow filtration, and affinity-based methods using conditioned cell culture media as the source. Isolated EVs were analyzed using mass spectrometry to characterize their proteomic profiles, and the shared protein content was evaluated across datasets. The gene ontology enrichment was further assessed using three bioinformatics platforms. Among the tested methods, ultracentrifugation emerged as the most effective isolation method in our analysis, thus underscoring its importance among commonly used techniques. Despite its analytical robustness, this method is unsuitable for routine clinical workflows due to its complexity and time demands. Our findings highlight the need for a standardized, less strenuous EV isolation method for clinical applications.
- extracellular vesicles,
- isolation methods,
- comparison,
- mass spectrometry,
- proteomic analysis
Citation: Klara Mrazova, Tomas Henek, Zuzana Mihal Jurikova, Simona Koznarova, Michaela Vasinova Galiova, Jaromir Bacovsky, Lenka Hernychova, Roman Hrstka. Comparative evaluation of five extracellular vesicle isolation methods using proteomic profiling[J]. AIMS Molecular Science, 2026, 13(1): 97-118. doi: 10.3934/molsci.2026006

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Abstract

Extracellular vesicles (EVs) research has gained a significant amount of attention in recent years. EVs are a heterogeneous group of different vesicles that vary in their origin, size, and function. The very nature of EVs, from their biogenesis to the contents of their cargo, offers many options for exploration. Despite being studied for a few decades now, there has not been an established standardized approach to isolation. However, the future use of EVs in clinical practice is conditioned by the optimization and standardization of isolation methods. In this study, we systematically compared common EV isolation techniques such as ultracentrifugation, precipitation, tangential flow filtration, and affinity-based methods using conditioned cell culture media as the source. Isolated EVs were analyzed using mass spectrometry to characterize their proteomic profiles, and the shared protein content was evaluated across datasets. The gene ontology enrichment was further assessed using three bioinformatics platforms. Among the tested methods, ultracentrifugation emerged as the most effective isolation method in our analysis, thus underscoring its importance among commonly used techniques. Despite its analytical robustness, this method is unsuitable for routine clinical workflows due to its complexity and time demands. Our findings highlight the need for a standardized, less strenuous EV isolation method for clinical applications.

Abbreviations

APOs

apoptotic bodies

biological process

cellular component

CCM

conditioned cell culture medium

DAVID

The Database for Annotation, Visualization and Integrated Discovery, DAVID Knowledgebase (v2023q4)

DLS

Dynamic light scattering

EDS

energy-dispersive X-ray spectroscopy

EVs

extracellular vesicles

Exo

exosomes

ExoEasy

exoEasy Maxi Kit

FBS

fetal bovine serum

FunRich

Functional Enrichment Analysis tool version 3.1.4

Gene Ontology knowledgebase version 2024-06-17

HCD

high-energy collision induced dissociation

LC-MS

liquid chromatography-mass spectrometry

MagCapture

MagCapture™ Exosome Isolation Kit PS Ver.2

molecular function

MVs

microvesicles

PDI

polydispersity index

PEG

polyethylene glycol

SEC

size exclusion chromatography

TEM

Transmission electron microscopy

TEI

Total Exosome Isolation (from cell culture media) isolation kit

TFF

tangential flow filtration

Tim4

T-cell immunoglobulin domain and mucin domain-containing protein 4

ultracentrifugation

XBP buffer

binding buffer

XE buffer

elution buffer

XWP buffer

wash buffer

Acknowledgments

This work was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU, by MH CZ - DRO (MMCI, 00209805) and by the project FCH-S-25-8807 of Ministry of Education, Youth and Sports of the Czech Republic.

Conflict of interest

The authors declare no conflict of interest in this paper.

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