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 chro...