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Proteomic Toolbox To Standardize the Separation of Extracellular Vesicles and Lipoprotein Particles
journal contribution
posted on 2018-08-06, 00:00 authored by Tingting Wang, Illarion V. TurkoCirculating 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.