Isolation and Proteomic Analysis of Mouse Serum Small Extracellular Vesicles for Individual Subject Analysis.

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: 30-150nm) 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 Longitudinal investigations of rodent models are challenging because of the limited amount of blood that can be withdrawn at each time; the American Association of Veterinary Science stipulates that fortnightly sampling should be limited to a maximum of 10% of the total blood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 75μL of serum. We developed an approach for the isolation and characterization of serum sEV proteins from just 50μL 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.