Core lipid, surface lipid and apolipoprotein composition analysis of lipoprotein particles as a function of particle size in one workflow integrating asymmetric flow field-flow fractionation and liquid chromatography-tandem mass spectrometry

Abstract

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.

Relation of High‐Density Lipoprotein Charge Heterogeneity, Cholesterol Efflux Capacity, and the Expression of High‐Density Lipoprotein‐Related Genes in Mononuclear Cells to the HDL‐Cholesterol Level

“…) despite the sixfold prevalence in the LDL particle content compared with VLDL. A very small level of apoA‐I in the VLDL fraction was reported also by others (Kuklenyik et al, ). Additionally, the level of apoA‐I‐containing small preβ1‐HDL in plasma (2–15% of total apoA‐I) (Nanjee and Brinton, ) is much higher compared with the apoA‐I fraction in VLDL even without counting the large preβ2‐HDL particles.…”

“…1) despite the sixfold prevalence in the LDL particle content compared with VLDL. A very small level of apoA-I in the VLDL fraction was reported also by others (Kuklenyik et al, 2018).…”

The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research

“…To address these issues, some investigators have turned to tangential field flow (TFF) (Heinemann et al, 2014) and asymmetric flow field-flow fractionation (A4F). A4F has been used to isolate EVs (Wagner et al, 2014;Zhang et al, 2018a) and LPPs (Kuklenyik et al, 2018;Rambaldi et al, 2009), as well as a novel class of nanoparticles termed exomeres (Zhang et al, 2018a) that have recently been shown to transfer functional cargo to recipient cells (Zhang and Coffey, 2019yy). Other methods for EV and LPP enrichment include immunoaffinity methods (including immunoprecipitation, immunoaffinity chromatography, protein capture microarrays, and microfluidic systems [Reá tegui et al, 2018y]), ion exchange sequential chromatography, and acoustofluidic separation (Wu et al, 2017).…”

HDL Dysfunctionality: Clinical Relevance of Quality Rather Than Quantity

“…High-density lipoproteins are one of the five major lipoproteins: chylomicrons, very low, intermediate, low, and high-density lipoproteins (VLDLs, IDLs, LDLs, and HDLs) classified by density [1]. In the basal state, HDL's functional properties, including antioxidant, anti-inflammatory, antiapoptotic, antithrombotic effects, contribute to its antiatherogenic role [2].…”