title: Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum.
abstract: The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAIL(WT)) and its death receptor 4 (DR4)-specific variant rhTRAIL(4C7) in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, (15)N-metabolically labeled internal standards of rhTRAIL(WT) and rhTRAIL(4C7) were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAIL(WT) and rhTRAIL(4C7) contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAIL(WT) and rhTRAIL(4C7) simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen , Antonius Deusinglaan 1, Groningen, 9713 AV The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wilffert
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Reis
authors.forename: Jos
authors.initials: J
authors.lastname: Hermans
authors.forename: Natalia
authors.initials: N
authors.lastname: Govorukhina
authors.forename: Tushar
authors.initials: T
authors.lastname: Tomar
authors.forename: Steven
authors.initials: S
authors.lastname: de Jong
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.forename: Nico C
authors.initials: NC
authors.lastname: van de Merbel
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008715:Methionine / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000097:blood*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000097:blood*; D011994:Recombinant Proteins / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011994:Recombinant Proteins; D008715:Methionine