Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

Abstract

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique. The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique. Lipoprotein particles are complex structures and the subject of several studies in which different functions have been attributed to both lipid (1.Wiesner P. Leidl K. Boettcher A. Schmitz G. Liebisch G. Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry.J. Lipid Res. 2009; 50: 574-585Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and associated protein (2.Davidsson P. Hulthe J. Fagerberg B. Camejo G. Proteomics of apolipoproteins and associated proteins from plasma high-density lipoproteins.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 156-163Crossref PubMed Scopus (71) Google Scholar, 3.Heinecke J.W. The HDL proteome: a marker–and perhaps mediator–of coronary artery disease.J. Lipid Res. 2009; 50: 167-171Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 4.Hoofnagle A.N. Heinecke J.W. Lipoproteomics: using mass spectrometry-based proteomics to explore the assembly, structure, and function of lipoproteins.J. Lipid Res. 2009; 50: 1967-1975Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 5.Ståhlman M. Davidsson P. Kanmert I. Rosengren B. Boren J. Fagerberg B. Camejo G. Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr.J. Lipid Res. 2008; 49: 481-490Abstract Full