High throughput quantification of apolipoproteins A-I and B-100 by isotope dilution MS targeting fast trypsin releasable peptides without reduction and alkylation

Authors

Bryan A. Parks, Centers for Disease Control and Prevention
David M. Schieltz, Centers for Disease Control and Prevention
Michael L. Andrews, Centers for Disease Control and Prevention
Michael S. Gardner, Centers for Disease Control and Prevention
John C. Rees, Centers for Disease Control and Prevention
Christopher A. Toth, Centers for Disease Control and Prevention
Jeffrey I. Jones, Centers for Disease Control and Prevention
Lisa G. McWilliams, Centers for Disease Control and Prevention
Zsuzsanna Kuklenyik, Centers for Disease Control and PreventionFollow
James L. Pirkle, Centers for Disease Control and Prevention
John R. Barr, Centers for Disease Control and Prevention

Date of this Version

2017

Document Type

Article

Citation

Proteomics Clin. Appl. 11, 7–8, 2017, 1600128, DOI 10.1002/prca.201600128.

Comments

U.S. government work.

Abstract

Purpose: 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.

Experimental design: 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.

Results: Themethod 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.

Conclusions and clinical relevance: 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.