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Updated: Apr 19, 2026
LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics
Published on: April 9, 2017
Targeting an acid labile aspartyl-prolyl amide bond as a viable alternative to trypsin digestion to generate a
Eliza N Fung1, Frank Zambito, Jonathan Haulenbeek
1R&D, Bristol-Myers Squibb Co., Route 206/Province Line Road, Lawrenceville, NJ 08543, USA.
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.
