Chapter title |
Isolating and Quantifying Plasma HDL Proteins by Sequential Density Gradient Ultracentrifugation and Targeted Proteomics.
|
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Chapter number | 7 |
Book title |
Quantitative Proteomics by Mass Spectrometry
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3524-6_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3522-2, 978-1-4939-3524-6
|
Authors |
Clark M. Henderson Ph.D., Tomas Vaisar Ph.D., Andrew N. Hoofnagle M.D., Ph.D., Clark M. Henderson, Tomas Vaisar, Andrew N. Hoofnagle |
Editors |
Salvatore Sechi |
Abstract |
The sensitivity and specificity of tandem mass spectrometers have made targeted proteomics the method of choice for the precise simultaneous measurement of many proteins in complex mixtures. Its application to the relative quantification of proteins in high-density lipoproteins (HDL) that have been purified from human plasma has revealed potential mechanisms to explain the atheroprotective effects of HDL. We describe a moderate throughput method for isolating HDL from human plasma that uses sequential density gradient ultracentrifugation, the traditional method of HDL purification, and subsequent trypsin digestion and nanoflow liquid chromatography-tandem mass spectrometry to quantify 38 proteins in the HDL fraction of human plasma. To control for the variability associated with digestion, matrix effects, and instrument performance, we normalize the signal from endogenous HDL protein-associated peptides liberated during trypsin digestion to the signal from peptides liberated from stable isotope-labeled apolipoprotein A-I spiked in as an internal standard prior to digestion. The method has good reproducibility and other desirable characteristics for preclinical research. |
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