Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles from Human Plasma: Enabling Single-Subject Analysis.

Federica Anastasi,Paolo Bongioanni,Maria Teresa Dell’Anno,Silvia Maria Masciandaro,Alessandra Falleni,Liam A Mcdonnell,Giovanni Signore,Renata Del Carratore

doi:10.3390/ijms22062951

Abstract

Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegenerative disorders. The possibility to isolate neuron-derived small extracellular vesicles (NDsEV) from blood represents a potential window into brain pathological processes. To date, the absence of sensitive NDsEV isolation and full proteome characterization methods has meant their protein content has been underexplored, particularly for individual patients. Here, we report a rapid method based on an immunoplate covalently coated with mouse monoclonal anti-L1CAM antibody for the isolation and the proteome characterization of plasma-NDsEV from individual Parkinson’s disease (PD) patients. We isolated round-shaped vesicles with morphological characteristics consistent with exosomes. On average, 349 ± 38 protein groups were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, 20 of which are annotated in the Human Protein Atlas as being highly expressed in the brain, and 213 were shared with a reference NDsEV dataset obtained from cultured human neurons. Moreover, this approach enabled the identification of 23 proteins belonging to the Parkinson disease KEGG pathway, as well as proteins previously reported as PD circulating biomarkers.

Highlights

Parkinson’s disease (PD) is a neurological disorder characterized by the progressive loss of nigral dopaminergic neurons and subsequent dopamine deficiency, which eventually results in motor symptoms and cognitive impairment [1,2]
Neurons obtained from neuroepithelial stem (NES) cells were differentiated to complete maturation as assessed by the expression of pan-neuronal markers (Figure S1) and media collected at the end of the differentiation process
Neuron-derived small extracellular vesicles from NES-cell-derived neurons (NESNDsEV) were isolated by Size Exclusion Chromatography (SEC) and sEV proteins analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS)

Summary

Introduction

Parkinson’s disease (PD) is a neurological disorder characterized by the progressive loss of nigral dopaminergic neurons and subsequent dopamine deficiency, which eventually results in motor symptoms and cognitive impairment [1,2]. It was reported that platelet contamination or variation in hemolysis could confound the reliable quantification of the blood Parkinson disease protein 7 and α-syn levels and further studies are needed to validate these proposed markers [9,10]. Small extracellular vesicles (sEV, diameter: 30–150 nm) are secreted by all cell types, are present in all body fluids, and carry molecules such as proteins, lipids, DNA, RNA, and miRNAs [11,12]. Their lipid bilayer of the outer membrane allows sEV to be stable for long periods of time and prevents degradation of their molecular content [13]. It has been demonstrated that sEV transport pathogenic proteins in PD [19], and their molecular content has been shown to correlate with PD onset and progression [20]. sEV of neuronal origin can be isolated using neuronal antigens, such as neural cell adhesion molecule

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Conclusion