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. (November 2014)

• Record Type: Journal Article
• Authors: Fung, Eliza N; Zambito, Frank; Haulenbeek, Jonathan; Piccoli, Steven P; Zhang, Yan; DeSilva, Binodh; Arnold, Mark; Kozhich, Alexander
• Is Part Of: Bioanalysis. Volume 6:Number 22(2014)
• Page Start: 2985
• Page End: 2998
• Publication Date: 2014-11
• Subjects: Drugs -- Analysis -- Periodicals; 615.19005
• DOI: 10.4155/bio.14.182
• Languages: English
• ISSNs: 1757-6180

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.