Abstract 391: Flow Field-flow Fractionation: a Powerful Underused Size Fractionation Technique for Composition Analysis of Lipoprotein Particles

Abstract

Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques for composition analysis of lipoproteins are limited by traditional preparative fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work a highly effective but underused size fractionation technique, asymmetric flow field-flow fractionation (AFFFF) was applied. We compared AFFFF with traditional techniques, including gel electrophoresis (GE), size exclusion chromatography (SEC), and gradient ultracentrifugation (GUC). Direct comparison of the techniques was performed by analysis of the same set of samples using sudan black staining. The AFFFF size separation allowed size resolution most similar to GE techniques, either tube gel or gradient gel. In addition, AFFFF and SEC was compared by collection of fractions where the average size in each fraction was measured by dynamic light scattering, and all major lipids and apolipoproteins were quantified by liquid chromatography tandem mass spectrometry methods (LC-MS/MS) in each fraction. The size resolution by AFFFF vs. SEC was compared based on the elution profiles of endogenous plasma proteins. On the hydrodynamic size scale, the half peak width of individual size species were 0.8-1 nm by AFFFF vs. 3-8 nm by SEC, showing the superior size resolution capability of the AFFFF technique. The gentle nature of the AFFFF separation relative to GUC was demonstrated by AFFFF separation of HDL sub-fractions with and without pre-fractionation by GUC, showing the significant loss of apoA-IV and apoCs, as well as a general shift of all lipid and protein profiles to smaller hydrodynamic size as a result of intense shearing forces and high salt conditions during GUC. Our results demonstrate that AFFFF coupled with LC-MS/MS deserves to be among the gold standard techniques for size and composition analysis of HDL, LDL and VLDL sub-classes.