Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry

Abstract

A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low-, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B-100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A-I and A-II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed-phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial elutions with acetonitrile. CE of the lipid-free apoprotein mixture shows the presence of apoC-I,II,III and apoE in the VLDL fraction, and apoA-I,II apoC-I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE-based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.