Proteomic Characterization of Human Plasma High Density Lipoprotein Fractionated by Gel Filtration Chromatography

Abstract

Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.

Structure of HDL: Particle Subclasses and Molecular Components

“…1). Importantly, these associations can in part be confirmed using an alternative approach of gel filtration subfractionation of HDL particles (Gordon et al 2010).…”

“…Thus, proteomics of apoA-I-containing fractions isolated from human plasma by a non-denaturing approach of fast protein liquid chromatography (FPLC) reveal the presence of up to 115 individual proteins per fraction, only up to 32 of which were identified as HDL-associated proteins in ultracentrifugally isolated HDL (Collins et al 2010). Indeed, co-elution with HDL of plasma proteins of matching size is inevitable in FPLC-based separation; the presence of a particular protein across a range of HDL-containing fractions of different size isolated by FPLC on the basis of their association with phospholipid would however suggest that such a protein is indeed associated with HDL (Gordon et al 2010). Remarkably, several of the most abundant plasma proteins, including albumin, haptoglobin and alpha-2-macroglobulin, are indeed present in all apoA-I-containing fractions isolated by FPLC (Collins et al 2010), suggesting their partial association with HDL by a non-specific, low-affinity binding.…”

The contribution of paraoxonase 1 and myeloperoxidase to HDL-cholesterol functionality

“…Thus, the structural variability of HDL-C makes it very sensitive to different metabolic signals which modify its structure and affect its functionality. In consequence, many authors have suggested that there is a great need for identifying the protein biomarkers for disturbances in the circulating HDL-C [25,41,95]. Recently, it has been proposed that among the proteins two enzymes -paraoxonase 1 (PON1) and myeloperoxidase (MPO) -may substantially affect and modify the HDL-C functionality.…”

Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers

“…The protein content of HDL particles has been characterized recently using various methods commonly described as proteomics. It has been shown that HDL2 fraction of CHD patients carries a distinct protein cargo (Vaisar et al 2010;Gordon et al 2010;Kavo et al 2012;Alwaili et al 2012). Proteomics as a new promising approach for detecting biomarkers and mechanisms could potentially explain the beneficial characteristics of HDL particles unrelated to their cholesterol content.…”

“…Proteomics as a new promising approach for detecting biomarkers and mechanisms could potentially explain the beneficial characteristics of HDL particles unrelated to their cholesterol content. To this end, novel sophisticated methods have been recently developed (Gordon et al 2010;Mazur et al 2010;Burillo et al 2013;Hoofnagle et al 2012;Mazur and Cardasis 2013;Riwanto et al 2013). High-throughput methods are urgently needed for large-scale epidemiological studies.…”