Simultaneous Isolation and Characterization of Lipoprotein Classes in Plasma, Including HDL Subclasses and the Uncharacterized Dense HDL

Lipoproteins (LPP) and extracellular vesicles are carriers of extracellular small RNA, with potential applications both in the areas of diagnostics and therapeutics. Lipid nanoparticles overlap across a range of densities and sizes in plasma, making them difficult to isolate intact and without contamination from other plasma components. Accurate characterization of their cargo through efficient isolation from other plasma components is required to understand their function. Here we describe the simultaneous separation of LPP classes using sequential flotation ultracentrifugation followed by size exclusion chromatography from 500{micro}L of starting plasma. Using western blot, denaturing and non-denaturing gel electrophoresis, nuclear magnetic resonance, and electron microscopy, we demonstrate separation of the LPP classes with minimal contamination. We also show unique lipidomic, proteomic and small RNA signatures for each LPP class, including, for the first time, very high density high density lipoprotein particles in the density range of 1.21-1.25 g/mL. MotivationIn plasma, lipid nanoparticles overlap in size and density, making it difficult to separate and isolate intact particles. Additionally, currently available methods do not allow for simultaneous characterization and isolation of these particles. This method resolves both challenges. The results presented also analyze and describe the presence of known lipoprotein particles, as well as those that have been previously under-characterized.