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.

Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non‐denaturing two‐dimensional gel electrophoresis

“…Spot 2 in Figure 1a has been identified as carboxylesterase, thus some lipids should be hydrolyzed by carboxylesterase on the PVDF membrane and so released from the HDL. It has been reported that lipids are removed from lipoproteins by treatment with chemicals such as bile salt sodium deoxycholate or sodium dodecyl sulfate (Macfarlane et al, 1997;Robern, 1982). These chemical treatments can impair enzymes activities, whereas enzymatic hydrolysis of lipids do not.…”

Investigation of a capillary electrophoretic approach for direct quantification of apolipoprotein A‐I in serum

“…This, as well as incomplete delipidation of the HDL particles [17,18] and/or comigrating serum components/proteins, may generate additional UV-absorbing peaks, possibly migrating close by or within the Apo A-I peak, thereby interfering the quantification of Apo A-I. Cruzado et al [17] and Macfarlane et al [18] demonstrated a double peak of Apo A-I based on incomplete delipidation of HDL. In our investigations, such interfering components have not been observed in the range of Apo A-I, neither in the 99 sera nor in the serum of a patient with Tangier disease, though in the "Tangier disease serum" all serum proteins (except Apo A-I), all kinds of lipids (released from delipidation of LDL and VLDL particles) and all other UVabsorbing substances are present.…”

“…Furthermore, the running buffer contributes also to the , 2 3 Focused on a simple sample preparation by dilution (running and sample preparation buffer are the same), SDS acts as the delipidating agent for the lipoprotein particles. But, as illustrated by MacFarlane et al [18], careful attention should be paid to the fact that lipids released in the delipidation process can associate with the surfactants in the running buffer, producing lipid-loaded micelles. This, as well as incomplete delipidation of the HDL particles [17,18] and/or comigrating serum components/proteins, may generate additional UV-absorbing peaks, possibly migrating close by or within the Apo A-I peak, thereby interfering the quantification of Apo A-I.…”

“…But, as illustrated by MacFarlane et al [18], careful attention should be paid to the fact that lipids released in the delipidation process can associate with the surfactants in the running buffer, producing lipid-loaded micelles. This, as well as incomplete delipidation of the HDL particles [17,18] and/or comigrating serum components/proteins, may generate additional UV-absorbing peaks, possibly migrating close by or within the Apo A-I peak, thereby interfering the quantification of Apo A-I. Cruzado et al [17] and Macfarlane et al [18] demonstrated a double peak of Apo A-I based on incomplete delipidation of HDL.…”

Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

“…Is reverse cholesterol transport, protection against oxidation, or some other process key to their effects? Clearly much remains to be learned about the structure and function of apolipoproteins arising from mutation, alternate splicing, processing, truncation, and deamidation [2][3][4].…”