title: 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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.
authors.affiliation: R&D, Bristol-Myers Squibb Co., Route 206/Province Line Road, Lawrenceville, NJ 08543, USA.
authors.forename: Eliza N
authors.initials: EN
authors.lastname: Fung
authors.forename: Frank
authors.initials: F
authors.lastname: Zambito
authors.forename: Jonathan
authors.initials: J
authors.lastname: Haulenbeek
authors.forename: Steven P
authors.initials: SP
authors.lastname: Piccoli
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Binodh
authors.initials: B
authors.lastname: DeSilva
authors.forename: Mark
authors.initials: M
authors.lastname: Arnold
authors.forename: Alexander
authors.initials: A
authors.lastname: Kozhich
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004151:Dipeptides / Q000737:chemistry*; D005346:Fibroblast Growth Factors / Q000737:chemistry* / Q000493:pharmacokinetics / Q000633:toxicity; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D000577:Amides; D004151:Dipeptides; C414620:fibroblast growth factor 21; C070752:aspartyl-proline; D005346:Fibroblast Growth Factors; D014357:Trypsin