Targeting an acid labile aspartyl–prolyl amide bond as a viable alternative to trypsin digestion to generate a surrogate peptide for LC–MS/MS analysis

Abstract

Background: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. Results & discussion: Taking advantage of a uniquely acid-labile aspartyl–prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200–50000 ng/ml. The assay accuracy and precision were between 92.8–99.8% and 3.9–14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. Conclusion: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.