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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.

Bioanalysis.. 2014-11;  6(22):2985-98
Fung EN, Zambito F, Haulenbeek J, Piccoli SP, Zhang Y, DeSilva B, Arnold M, Kozhich A. R&D, Bristol-Myers Squibb Co., Route 206/Province Line Road, Lawrenceville, NJ 08543, USA.
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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 analy... More

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