Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple- Reaction–Monitoring Mass Spectrometry

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 56; no. 12; pp. 1804 - 1813
• Main Authors: Agger, Sean A, Marney, Luke C, Hoofnagle, Andrew N
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 01.12.2010; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Apolipoprotein A-I - blood; Apolipoproteins; Apolipoproteins B - blood; Biological and medical sciences; Biomarkers; Biomarkers - blood; Calibration; Chromatography; Chromatography, High Pressure Liquid; Fundamental and applied biological sciences. Psychology; Human subjects; Humans; Immunoassay; Immunoassays; Investigative techniques, diagnostic techniques (general aspects); Ionization; Isotopes; Mass spectrometry; Medical sciences; Molecular biophysics; Nephelometry and Turbidimetry; Peptides; Plasma; Protein Denaturation; Proteins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Studies; Tandem Mass Spectrometry; Trifluoroethanol; Urea; Apolipoprotein B; Multiple; Surveillance; Clinical biology; Apolipoprotein AI; Quantization; Biochemistry; Liquid chromatography; Molecular biology; Reaction; Mass spectrometry; Quantitative analysis
• ISSN: 0009-9147; 1530-8561; 1530-8561
• DOI: 10.1373/clinchem.2010.152264

If liquid-chromatography-multiple-reaction-monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay - 36.6; S(x|y) = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; S(x|y) = 7.9 for apoB). Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.