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Abstract
Background: We describe an antibody-free approach to quantify rhTRAILWT (wild-type) and its closely related death receptor 4 selective variant rhTRAIL4C7 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 rhTRAILWT (8.5 pM) and 2 ng/ml for rhTRAIL4C7 (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.
Supplementary Data
Financial & competing interests disclosure
The Dutch Technology Foundation (STW) is gratefully acknowledged for financial support (grant 11056). Further financial support was provided by PRA Health Sciences (Assen, the Netherlands), Spark Holland (Emmen, The Netherlands), Merck Sharp & Dohme (MSD, Oss, The Netherlands), TOSOH Bioscience (Stuttgart, Germany), Beckman Coulter Inc. (IN, ISAs) and Bionavis (Tampere, Finland) as part of this grant. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.