title: Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics.
abstract: High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using (15)N-labeled apolipoprotein A-I (HDL's most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies.
  
  
  CONCLUSIONS
  HDL, a complex matrix composed of lipids and proteins, is implicated in cardioprotection. Its cholesterol content correlates inversely with cardiovascular disease and it is the current metric to assess cardiovascular risk. However, the cholesterol content does not capture HDL's complexity and heterogeneity. Devising metrics that better capture HDL's cardioprotective effects, we developed an optimized method for quantification of HDL proteome, using PRM in concert with a single labeled protein as internal standard. The availability of a method that increases sample throughput without compromising the reproducibility, sensitivity, and accuracy could therefore point to better risk assessment for CVD or other diseases.
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA. Electronic address: ronsein@uw.edu.
authors.forename: Graziella E
authors.initials: GE
authors.lastname: Ronsein
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Pamir
authors.affiliation: Genome Sciences, University of Washington, Seattle, WA 98109, USA.
authors.forename: Priska D
authors.initials: PD
authors.lastname: von Haller
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA; Genome Sciences, University of Washington, Seattle, WA 98109, USA.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Oda
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA; Genome Sciences, University of Washington, Seattle, WA 98109, USA.
authors.forename: Gail P
authors.initials: GP
authors.lastname: Jarvik
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA.
authors.forename: Tomas
authors.initials: T
authors.lastname: Vaisar
authors.affiliation: Departments of Medicine, University of Washington, Seattle, WA 98109, USA.
authors.forename: Jay W
authors.initials: JW
authors.lastname: Heinecke
mesh_terms: D016632:Apolipoprotein A-I / Q000032:analysis / Q000737:chemistry; D005260:Female; D006801:Humans; D008075:Lipoproteins, HDL / Q000032:analysis / Q000097:blood*; D008297:Male; D008991:Monitoring, Physiologic / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D009587:Nitrogen Isotopes
keywords: HDL; PRM; SRM; Single internal standard protein; Targeted proteomics