Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.
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.
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.
Abhinav
A
Kumar
Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Bevin
B
Gangadharan
Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Nicole
N
Zitzmann
D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000097:blood* / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D002138:Calibration; D006031:Glycosylation; D006801:Humans; D057230:Limit of Detection; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D001053:Apolipoproteins; D010455:Peptides; C036321:apolipoprotein F; D014357:Trypsin
Apolipoprotein F; Liquid chromatography–mass spectrometry; Liver fibrosis; Multiple reaction monitoring; Multiple reaction monitoring cubed; Skyline