Abstract
Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.
Highlights
Extracellular vesicles (EVs) comprise a heterogeneous class of nanosized membrane vesicles released from all cell types and play an essential role in intercellular communication [1]
Instead of total proteome analysis, we focused on a small group of rigorously selected proteins associated with either plasma or endosomal membrane and used targeted proteome analysis based on multiple reaction monitoring (MRM) mass spectrometry [20,21] to quantify these proteins in preparations of microvesicles and exosomes
Since absolute quantifications of GP Ib beta, EH domain-containing protein 4 (EHD4), and cytochrome P-450 5A1 (CYP5A1) were essentially identical based on individual peptides, we have concluded that cell EVs in our preparations are not contaminated with bovine EVs
Summary
Extracellular vesicles (EVs) comprise a heterogeneous class of nanosized membrane vesicles released from all cell types and play an essential role in intercellular communication [1]. More than a dozen terms based on biogenesis and size have been used to define EVs [8] This long and confusing list was recently narrowed to a few major groups, including microvesicles and exosomes as the most common [9]. Proteomes 2020, 8, 33 exosomes are small, these EVs have commonly been separated by differential ultracentrifugation, where the particle fraction obtained by centrifugation (up to 20,000× gn ) is called microvesicles. Instead of total proteome analysis, we focused on a small group of rigorously selected proteins associated with either plasma or endosomal membrane and used targeted proteome analysis based on multiple reaction monitoring (MRM) mass spectrometry [20,21] to quantify these proteins in preparations of microvesicles and exosomes. The proteomic patterns of microvesicle and exosome preparations permitted us to evaluate their membrane origin
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