title: Isolating and Quantifying Plasma HDL Proteins by Sequential Density Gradient Ultracentrifugation and Targeted Proteomics.
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.
authors: Department of Laboratory Medicine, University of Washington School of Medicine, Box 357110, Seattle, WA, 98195-7110, USA.
authors: Clark M
authors: CM
authors: Henderson
authors: Department of Medicine, University of Washington School of Medicine, Box 358055, Seattle, WA, 98195-8055, USA.
authors: Tomas
authors: T
authors: Vaisar
authors: Department of Laboratory Medicine, University of Washington School of Medicine, Box 357110, Seattle, WA, 98195-7110, USA. ahoof@u.washington.edu.|Department of Medicine, University of Washington School of Medicine, Box 358055, Seattle, WA, 98195-8055, USA. ahoof@u.washington.edu.
authors: Andrew N
authors: AN
authors: Hoofnagle
mesh_terms: D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D008075:Lipoproteins, HDL / Q000032:analysis* / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins; D008075:Lipoproteins, HDL; D010455:Peptides
keywords: Density gradient ultracentrifugation; High-density lipoprotein; Multiple reaction monitoring; Nanoflow liquid chromatography; Protein quantification; Quantitative proteomics; Skyline; Stable isotope-labeled internal standard protein; Tandem mass spectrometry