title: Highly sensitive antibody-free μLC-MS/MS quantification of rhTRAIL in serum.
abstract: BACKGROUND
  We describe an antibody-free approach to quantify rhTRAIL(WT) (wild-type) and its closely related death receptor 4 selective variant rhTRAIL(4C7) in human and murine serum by multiplex LC-MS/MS on a microfluidics interface.
  
  
  METHODS
  Enrichment of rhTRAIL was performed by strong cation-exchange (SCX) followed by immobilized metal affinity (IMAC) solid-phase extraction. This was followed by trypsin digestion and using methionine-containing signature peptides after fully oxidizing the methionine residue with 0.25% (w/w) hydrogen peroxide.
  
  
  CONCLUSIONS
  Absolute quantification was reaching down to 0.5 ng/ml for rhTRAIL(WT) (8.5 pM) and 2 ng/ml for rhTRAIL(4C7) (34 pM) in 100 μl human serum. To support preclinical studies in mice, the analysis was optimized further, for a sample volume of 20 μl murine serum.
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wilffert
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Angela
authors.initials: A
authors.lastname: Asselman
authors.affiliation: Department of Pathology, University of Pisa, Via Roma 55, 56126 Pisa, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Donzelli
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Hermans
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Natalia
authors.initials: N
authors.lastname: Govorukhina
authors.affiliation: Pharmaceutical Biology, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.|PRA Health Sciences, Bioanalytical Laboratory, Amerikaweg 18, 9407 TK Assen, The Netherlands.
authors.forename: Nico C
authors.initials: NC
authors.lastname: van de Merbel
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D056656:Lab-On-A-Chip Devices; D057230:Limit of Detection; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000097:blood; D052616:Solid Phase Extraction / Q000379:methods*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human
keywords: 15N-metabolically labeled internal standard; TNF-related apoptosis-inducing ligand (TRAIL); antibody-free; immobilized metal affinity (IMAC) enrichment; quantitative LC–MS/MS; strong cation exchange (SCX) enrichment