Identification of metabolites using high resolution MSn: novel tools for data acquisition and spectral tree interpretation

Metabolite identification is one of the central aspects of metabolomics and one of the major bottlenecks prohibiting biological interpretation of results obtained from quantitative studies. The strategy for metabolite identification developed in the project is centred around multistage mass spectrometry.
An acquisition method yielding reproducible and comprehensive fragmentation data that are highly characteristic for metabolite structures was developed. Several acquisition parameters were optimised. The reproducibility of the acquisition was verified by results of inter-laboratory test (in Leiden, Wageningen, Delft and Zeist). The method is successfully used in DCL Leiden, PRI Wageningen and in DSM Delft.
In close collaboration with project MI2, the procedure for processing and analysing the obtained MSn data was developed and implemented. The information of the fragmentation of metabolite standards is cleaned from spectral artefacts and stored as fragmentation trees in the database. The fragmentation trees are highly characteristic for the metabolite structure and allow differentiation of structural isomers (as demonstrated on isomeric prostaglandins E2 and D2). The specificity was successfully demonstrated over a wide concentration range of model metabolites showing the robustness of the method. The database of the metabolite fragmentation trees is being build and currently consists of the results of 1600 MSn experiments performed for 432 human metabolite standards both in positive and negative ionisation mode.
The protocol for MSn acquisition of metabolites in biological samples was developed and implemented to analyse human urine. The tools were tested in the early stage in assisting the process of manual identification of potential biomarkers of diabetic nephropathy. In close collaboration with other MI projects, the follow-up study will demonstrate developed tools in the integrated metabolite identification pipeline.