Proteomic Toolbox To Standardize the Separation of Extracellular Vesicles and Lipoprotein Particles

Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and density, traditional approaches to separate EVs and LPs often fail to provide the required resolution. Further development and standardization of affinity-based protocols is necessary, and a quantitative method is needed to assess the efficiency of LP depletion from EV samples. In the present study, we propose the simultaneous quantification of three groups of proteins by mass spectrometry as a toolbox to evaluate prospective separation protocols. We generated ¹⁵N-labeled internal standards for quantification of (i) EV-specific proteins, (ii) all classes and subclasses of apolipoproteins constituting LPs, and (iii) several major serum proteins. These standards were then used in multiple reaction monitoring assay to evaluate the performance of size-exclusion chromatography, heparin-Sepharose, lipopolysaccharide-Sepharose, (2-hydroxypropyl)-β-cyclodextrin-Sepharose, and concanavalin A-Sepharose in separating serum EVs and LPs. The efficiency of a resin to separate EVs from non-EV substances could be jeopardized by simultaneous EV aggregation. Therefore, dynamic light scattering analysis was used in this study in addition to the proteomic toolbox when making a recommendation to use particular resin for EV isolation. On the basis of our measurements, we concluded that none of the individual separation protocols used in this study resulted in LP-free EVs, and the combination of two protocols may be complex due to low EV yield. Overall, this further points to the importance of proposed proteomic toolbox for the future evaluation of EV separation protocols.