Highly sensitive antibody-free μLC-MS/MS quantification of rhTRAIL in serum
Daniel Wilffert1, Angela Asselman1, Riccardo Donzelli2
1Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
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.
Methodology:
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.
Conclusion:
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.
