PhD defence Daniel Wilffert
|When:||Fr 26-02-2016 at 14:30|
Antibody-free LC-MS/MS protein analysis of TRAIL
The central theme of the thesis of Daniel Wilffert is the development of highly sensitive liquid chromatography - mass spectrometry (LC-MS/MS) assays for protein quantification in biological matrices without the use of antibodies.
A literature review in chapter 2 discusses the wide range of possibilities for antibody-free workflows that may be applied prior to LC-MS/MS protein quantification. In chapter 3 we describe an antibody-free LC-MS/MS method that is able to discriminate the closely related DR4-specific variant rhTRAIL4C7 from wild type rhTRAIL and to measure both forms simultaneously with a lower limit of quantitation of 20 ng/mL (350 pM) in human or mouse serum. The method was fully validated according to international guidelines and its suitability was proven by analysis of the pharmacokinetic profiles of both variants, which were simultaneously dosed to mice.
In chapter 4, a method is presented in which IMAC enrichment is extended with an initial strong cation exchange (SCX) SPE step and in which the LC separation is miniaturized using a microfluidic LC-MS interface. This provides an improved sample clean-up and, consequently, an increase in sensitivity in human serum down to 0.5 ng/mL (8.8 pM) for rhTRAILWT, which is close to the LLOQ of 0.2 ng/mL (3.5 pM) for a validated ELISA that was used in Phase-I clinical studies.
In chapter 5, SCX enrichment was used at the peptide instead of at the protein level. This allowed, for the first time, to quantify sTRAIL at endogenous levels by LC-MS/MS down to a level of 3 pM in sputum and saliva.
Promotores: Prof.dr. R.P.H. Bischoff, Prof.dr. N.C. van de Merbel en Prof.dr. W.J. Quax