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Isolating and Quantifying Plasma HDL Proteins by Sequential Density Gradient Ultracentrifugation and Targeted Proteomics

  • Clark M. Henderson
  • Tomas Vaisar
  • Andrew N. HoofnagleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1410)

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.

Key words

Multiple reaction monitoring Tandem mass spectrometry Nanoflow liquid chromatography Stable isotope-labeled internal standard protein Skyline High-density lipoprotein Protein quantification Quantitative proteomics Density gradient ultracentrifugation 

Notes

Acknowledgements

This work was supported by NIH grants: HL111375, DK035816, HL089504 and NIH training grant T32HL007028 and AHA grants 0830231N and 14GRNT18410022. We thank Jennifer Wallace for her contributions to this chapter.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Clark M. Henderson
    • 1
  • Tomas Vaisar
    • 2
  • Andrew N. Hoofnagle
    • 1
    • 2
    Email author
  1. 1.Department of Laboratory MedicineUniversity of Washington School of MedicineSeattleUSA
  2. 2.Department of MedicineUniversity of Washington School of MedicineSeattleUSA

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