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J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Oct 15;1033-1034:278-286. doi: 10.1016/j.jchromb.2016.08.038. Epub 2016 Aug 27.

Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

Author information

1
Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom. Electronic address: abhinav.kumar@bioch.ox.ac.uk.
2
Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

Abstract

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.

KEYWORDS:

Apolipoprotein F; Liquid chromatography–mass spectrometry; Liver fibrosis; Multiple reaction monitoring; Multiple reaction monitoring cubed; Skyline

PMID:
27592286
DOI:
10.1016/j.jchromb.2016.08.038
[Indexed for MEDLINE]

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