title: High throughput quantification of apolipoproteins A-I and B-100 by isotope dilution MS targeting fast trypsin releasable peptides without reduction and alkylation.
abstract: Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form.
  
  
  
  A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis.
  
  
  
  The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels.
  
  
  
  The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Parks
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Schieltz
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Andrews
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gardner
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Jon C
authors.initials: JC
authors.lastname: Rees
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Toth
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Jones
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Lisa G
authors.initials: LG
authors.lastname: McWilliams
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Kuklenyik
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.affiliation: Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism; D053299:Apolipoprotein B-100 / Q000737:chemistry* / Q000378:metabolism; D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D002853:Chromatography, Liquid; D006801:Humans; D007554:Isotopes / Q000737:chemistry; D016014:Linear Models; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism*
chemical_list: D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D007554:Isotopes; D010446:Peptide Fragments; D014357:Trypsin
keywords: Apolipoproteins; Cardiovascular system; LC-MS/MS; Mass Spectrometry; Quantitation