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

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 o...

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Bibliographic Details
Published in:	Scientific reports Vol. 11; no. 1; p. 16086
Main Authors:	Zheng, Jack Jingyuan, Agus, Joanne K., Hong, Brian V., Tang, Xinyu, Rhodes, Christopher H., Houts, Hannah E., Zhu, Chenghao, Kang, Jea Woo, Wong, Maurice, Xie, Yixuan, Lebrilla, Carlito B., Mallick, Emily, Witwer, Kenneth W., Zivkovic, Angela M.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 09-08-2021 Nature Publishing Group Nature Portfolio
Subjects:	631/1647/2067 631/1647/2196 631/1647/2230 631/1647/2258 631/1647/296 Acyltransferase Cholesterol Chromatography Chromatography, Gel - methods Chromatography, Liquid - methods Clusterin Electrophoresis, Polyacrylamide Gel - methods Flotation Gel electrophoresis High density lipoprotein Humanities and Social Sciences Humans Lecithin Lipoproteins, HDL - blood Lipoproteins, HDL - isolation & purification Liquid chromatography multidisciplinary Particle Size Phospholipid transfer protein Phospholipids Plasma proteins Polyacrylamide Proteins Proteins - isolation & purification Proteomics Science Science (multidisciplinary) Structure-function relationships Transmission electron microscopy Ultracentrifugation Ultracentrifugation - methods Vitamin D
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-021-95451-3