title: Reagent-free LC-MS/MS-based pharmacokinetic quantification of polyhistidine-tagged therapeutic proteins.
abstract: OBJECTIVE
  Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS.
  
  
  RESULTS
  A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml.
  
  
  CONCLUSIONS
  The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Jianxia
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Jing Man
authors.initials: JM
authors.lastname: Wong
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Dickerson
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Hall
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Dan A
authors.initials: DA
authors.lastname: Rock
authors.affiliation: Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Carlson
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D006639:Histidine / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D051379:Mice; D008819:Mice, Nude; D010446:Peptide Fragments / Q000032:analysis*; D051381:Rats; D011994:Recombinant Proteins / Q000493:pharmacokinetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014018:Tissue Distribution
chemical_list: D008670:Metals; D010446:Peptide Fragments; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine
keywords: LC–MS/MS; immobilized metal ion affinity chromatography; polyhistidine-tagged proteins; reagent-free workflow; signature peptides; stable isotope-labeled internal standard