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

• Liver fibrosis biomarker assay by LC–MS.
• Absolute quantification of APO-F in human plasma by MRM.
• Peptide selection and library creation using Skyline for MRM.
• Reduction of interference by MRM3.
• Presence of glycopeptide by MRM and Enhanced product ion scanning.

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 MRM3) 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 445.2 ng/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.