Comprehensive account of exosome isolation from rat substantia nigra for mass spectrometry-based proteomics study

Exosomes, small extracellular vesicles originating from endocytic processes, have garnered increasing attention due to their roles in both physiological functions and pathological conditions. Initially identified in the 1980 s, exosomes are formed within multivesicular bodies (MVBs) through the invagination of the endosomal membrane, leading to the creation of intraluminal vesicles (ILVs). These ILVs can either be degraded by lysosomes or released into the extracellular space as exosomes, facilitating intercellular communication. In the nervous system, exosomes are implicated in various functions, including neural development and the progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. This study presents a novel protocol for the isolation and proteomic analysis of exosomes derived from the substantia nigra (SN) of rat brains. By employing a combination of differential centrifugation and immunocapture techniques, we achieved a purer exosome fraction and higher exosome yield compared to traditional ultracentrifugation methods. Our proteomics analysis identified 51, 48, and 70 proteins from three distinct exosome samples (SN-EV-1, SN-EV-2, and SN-EV-4), with Gene Ontology annotation revealing their involvement in diverse biological functions. This research not only establishes a reliable method for isolating brain-derived exosomes but also sets the stage for comparative studies between healthy and neurodegenerative conditions. Ultimately, our findings aim to enhance the understanding of exosomal roles in disease mechanisms and contribute to the identification of potential biomarkers and therapeutic targets for neurodegenerative disorders.