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Updated: Sep 25, 2025
Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies
Published on: January 31, 2025
Isolation and Proteomic Analysis of Mouse Serum Small Extracellular Vesicles for Individual Subject Analysis
Federica Anastasi1,2, Marialaura Dilillo2, Davide Pellegrini1,2
1NEST Laboratories, Scuola Normale Superiore, Pisa, Italy.
Abstract:
Proteomics characterization of blood and circulating material has been extensively explored for the study of pathological states. In particular, circulating small extracellular vesicles (sEV, diameter: 30-150 nm) are known to play an important role in intercellular communication processes, and proteomics profiling has been explored to develop minimally invasive assays for disease monitoring and diagnosis. Due to the genetic and physiological similarities between the two species, and also on account of their shorter life span and rapid disease progression, rodent models are the most commonly used animal model for many human diseases. Such models have provided invaluable insight into the molecular mechanisms of disease progression, candidate drug efficacy, therapy monitoring, and biomarkers research.Longitudinal investigations, in which individuals are monitored over periods of time, are more able to resolve molecular changes during disease progression because they circumvent the inter-individual variation. Longitudinal investigations of rodent models are challenging because of the limited amount of blood that can be withdrawn at each time; the American Association of Veterinary Science stipulates that fortnightly sampling should be limited to a maximum of 10% of the total blood volume. For adult mice this corresponds to approximately 75 μL of serum. We developed an approach for the isolation and characterization of serum sEV proteins from just 50 μL of serum, for longitudinal studies of disease mouse models. This chapter describes in detail the steps and considerations involved in the sEV isolation, morphological characterization, and proteome profiling by mass spectrometry.
Insights
We developed a method to analyze proteins from small extracellular vesicles (sEVs) in just 50 μL of mouse serum. This technique enables longitudinal studies of disease progression in rodent models using minimal blood volumes.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomedical Research
Background:
- Circulating small extracellular vesicles (sEVs) are crucial for intercellular communication and are studied for disease monitoring via proteomics.
- Rodent models are vital for human disease research due to genetic similarities and shorter lifespans.
- Longitudinal studies in rodents are challenged by limited blood withdrawal volumes (approx. 75 μL serum per sample).
Purpose of the Study:
- To develop a method for isolating and characterizing serum sEV proteins from small blood volumes (50 μL) for longitudinal studies in mouse models.
- To enable detailed proteomic profiling of sEVs from limited serum samples, facilitating disease progression research.
Main Methods:
- Developed a protocol for sEV isolation from 50 μL of mouse serum.
- Performed morphological characterization of isolated sEVs.
- Conducted proteome profiling of sEVs using mass spectrometry.
Main Results:
- Successfully isolated and characterized serum sEVs from a minimal blood volume.
- Established a method for proteomic analysis of sEVs suitable for longitudinal studies.
- Provided detailed steps and considerations for sEV isolation, characterization, and proteomic profiling.
Conclusions:
- The developed approach allows for longitudinal proteomic analysis of serum sEVs in rodent models using significantly reduced blood volumes.
- This method overcomes a key limitation in rodent studies, enabling more robust research into disease mechanisms and biomarker discovery.
- The described protocol facilitates minimally invasive, in-depth investigation of sEVs in disease progression and therapeutic monitoring.
