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Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation
Published on: January 28, 2021
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Isolating and Quantifying Plasma HDL Proteins by Sequential Density Gradient Ultracentrifugation and Targeted
Clark M Henderson1, Tomas Vaisar2, Andrew N Hoofnagle3,4
1Department of Laboratory Medicine, University of Washington School of Medicine, Box 357110, Seattle, WA, 98195-7110, USA.
Methods in Molecular Biology (Clifton, N.J.)
|February 13, 2016
Summary
Targeted proteomics precisely quantifies HDL proteins in human plasma. This method reveals potential mechanisms behind high-density lipoprotein
Area of Science:
- Proteomics
- Biochemistry
- Cardiovascular Research
Background:
- Targeted proteomics offers high sensitivity and specificity for protein quantification.
- High-density lipoproteins (HDL) play a role in atheroprotection.
- Understanding HDL protein composition is key to elucidating its protective effects.
Purpose of the Study:
- To develop and validate a moderate-throughput method for quantifying proteins within human plasma HDL.
- To identify potential mechanisms underlying the atheroprotective properties of HDL.
Main Methods:
- Isolation of HDL from human plasma via sequential density gradient ultracentrifugation.
- Trypsin digestion of HDL proteins followed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Normalization using stable isotope-labeled apolipoprotein A-I as an internal standard to control for analytical variability.
Main Results:
- Quantification of 38 distinct proteins within the purified HDL fraction.
- Demonstrated good reproducibility of the developed method.
- Established a reliable approach for preclinical research applications.
Conclusions:
- The described targeted proteomics method enables precise quantification of HDL proteins.
- This technique provides insights into the atheroprotective mechanisms of HDL.
- The method is suitable for reproducible preclinical investigations.
