Abstract
The multiple roles of extracellular vesicles (EVs) in pathogenesis have received much attention, as they are valuable as diagnostic and prognostic biomarkers. We employed polymeric EV precipitation to isolate EVs from clinical specimens to overcome the limitations of ultracentrifugation (UC), such as low protein yields, a large volume of specimens used, and time requirements. Multiple-cycle polymeric EV precipitation was applied to enhance the purity of the EV fractions with a small sample volume. Then, the purity was assessed using a multiple reaction monitoring (MRM) panel consisting of alpha-2-macroglobulin (A2M), thrombospondin 1 (THBS 1), galectin 3 binding protein (LGALS3BP), and serum albumin (ALB). For purity evaluation, the EV fractions isolated from blood specimens were subjected to shotgun proteomics and MRM-based targeted proteomics analyses. We demonstrate that the modified method is an easy and convenient method compared with UC. In the shotgun proteomics analysis, the proteome profile of EV fraction contains 89% EV-related proteins by matching with the EVpedia database. In conclusion, we suggest that multiple-cycle polymeric EV precipitation is not only a more effective method for EV isolation for further proteomics-based experiments, but may also be useful for further clinical applications due to the higher EV yield and low sample requirements.
Highlights
Extracellular vesicles (EVs) are spherical cell-derived membrane particles classified into exosomes (30–120 nm), microvesicles (100–1000 nm), and apoptotic bodies (800–5000 nm) according to cellular origin, function, and biogenesis [1,2]
extracellular vesicles (EVs) are intracellular hormone-like messengers such as protein carriers or RNA carriers that are reported to be deeply involved in diseases and biological processes such as immune response regulation, blood coagulation, cell migration, cell differentiation, and cell-to-cell communication [4,5,6,7,8]
Differences in protein concentrations between purified and nonpurified EV fractions were assessed by the nonparametric Wilcoxon test; p ≤ 0.05 was considered statistically significant. This is the first report highlighting the usefulness of multiple-cycle polymeric EV precipitation and an multiple reaction monitoring (MRM) panel for purity evaluation
Summary
Extracellular vesicles (EVs) are spherical cell-derived membrane particles classified into exosomes (30–120 nm), microvesicles (100–1000 nm), and apoptotic bodies EVs are intracellular hormone-like messengers such as protein carriers or RNA carriers that are reported to be deeply involved in diseases and biological processes such as immune response regulation, blood coagulation, cell migration, cell differentiation, and cell-to-cell communication [4,5,6,7,8] These vesicles are highly released in diseases including cancer and play a role in spreading proteins that cause disease to other cells [9]. We present multiple-cycle polymeric EV precipitation with high purity, which may be applicable in clinical practice and provides EV marker proteins for purity evaluation. This is the first report on the usefulness of multi-cycle polymer-based reagent treatment compared with multi-cycle UC [23], including quantitative results of each EV fraction acquired from a different reagent processing cycle and verified by MRM assay
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