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Updated: Jun 10, 2026
Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation
Published on: January 28, 2021
Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography
Scott M Gordon1, Jingyuan Deng, L Jason Lu
1Center for Lipid and Arteriosclerosis Science, University of Cincinnati, Cincinnati, Ohio 45237-0507, USA. Gordonst@mail.uc.edu
Insights
High-density lipoprotein (HDL) cholesterol levels protect against cardiovascular disease. This study reveals new HDL-associated proteins, expanding our understanding of HDL
Area of Science:
- Proteomics
- Lipid Metabolism
- Cardiovascular Disease Research
Background:
- Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with cardiovascular disease incidence.
- HDL particles contain over 50 proteins, with newly identified ones suggesting roles beyond lipid transport in immunity and inflammation.
- Traditional ultracentrifugation methods for HDL isolation may disrupt protein interactions and alter particle function.
Purpose of the Study:
- To develop and apply a novel method for HDL fractionation and proteomic analysis.
- To identify novel HDL-associated proteins and characterize HDL heterogeneity.
- To investigate the functional implications of identified HDL proteins in complement regulation and protease inhibition.
Main Methods:
- High-resolution size exclusion chromatography was used to fractionate normal human plasma into 17 phospholipid-containing subfractions.
- Phospholipid binding resin and electrospray ionization mass spectrometry were employed to identify associated proteins.
- Analysis focused on protein distribution across different lipoprotein particle sizes.
Main Results:
- Fourteen new phospholipid-associated proteins were identified that co-migrate with HDL.
- Several identified proteins are involved in complement regulation and protease inhibition, supporting novel HDL functions.
- Unprecedented resolution revealed significant heterogeneity in HDL protein distribution across particle sizes, suggesting distinct subpopulations.
Conclusions:
- The novel fractionation and proteomic approach provides a more accurate view of HDL composition and function.
- The identification of new HDL-associated proteins expands the known roles of HDL in innate immunity and inflammation.
- Observed HDL heterogeneity indicates the existence of functionally distinct HDL particle subpopulations.
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.
