Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

Abstract

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

High-Density Lipoprotein Changes in Alzheimer’s Disease Are APOE Genotype-Specific

“…HDL particles from plasma were isolated by two-step sequential flotation density-ultracentrifugation, followed by size exclusion chromatography, to yield highly purified HDL fractions, as previously described [ 13 ]. Following the manufacturer’s instructions, the total HDL protein was measured using a Micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA, catalog 23235).…”

“…The software ImageJ [ 17 ] was used to characterize the HDL particle size from the TEM micrographs, following a previously published procedure [ 13 ], with minor modifications: Noise in the micrographs was first removed using the “Bandpass Filter” function, with “filter large = 100, filter small = 10, suppress = None, and tolerance = 5 autoscale saturate” parameters. The contrast of the cleaned micrograph was then set to “min = 50, max = 205”.…”

Quantitative glycoproteomics of high-density lipoproteins

“… 26 Purified HDL particles were isolated through a two-step HDL isolation method which isolates HDL particles first by density using sequential flotation ultracentrifugation followed by size exclusion chromatography, as described previously. 14 Briefly, 500 μL of plasma was underlaid under KBr solution at a density of 1.0060 g mL −1 to remove triglyceride-rich, low density (<1.006 g mL −1 ) particles, including chylomicrons and VLDL, and submitted to ultracentrifugation in an Optima MAX-TL Ultracentrifuge with (Beckman-Coulter) fixed angle rotor at 110 000 RPM and 14 °C for 30 minutes. After centrifugation, the supernatant was removed by aspiration, and the remaining fraction containing HDL, LDL, albumin, and plasma proteins was adjusted to a density of 1.210 g mL −1 with 1.340 g mL −1 KBr solution and underlaid under clean 1.210 g mL −1 density solution, then submitted to ultracentrifugation at 110 000 RPM and 14 °C for 3 hours and 30 minutes.…”

“…Blood samples were collected before and after four weeks of supplementation. HDL was isolated using an optimized, validated method, 14 and peptides and glycopeptides were quantified using the new workflow.…”

Lipid-Based Nutrient Supplementation Increases High-Density Lipoprotein (HDL) Cholesterol Efflux Capacity and Is Associated with Changes in the HDL Glycoproteome in Children

“…HDL Isolation. HDL particles were isolated through a twostep HDL isolation method modified from the study by Holzer et al 44 which isolates HDL particles first by density using sequential flotation ultracentrifugation as previously described 45 followed by fast protein liquid chromatography (FPLC). Briefly, 500 μL of plasma was underlaid under KBr solution at a density of 1.006 g/mL to remove triglyceride-rich, low-density (<1.006 g/mL) particles, including chylomicrons and very low-density lipoproteins, and subjected to ultracentrifugation in an Optima MAX-TL Ultracentrifuge with (Beckmann-Coulter) fixed angle rotor at 110,000 rpm and 14 °C for 30 min.…”