This project aims at using the latest technological improvements to develop a robust state-of-the art bioanalytical platform that can be applied in metabolomics research. The project is divided into three work packages (WPs):
WP1: Instrumental detection of metabolome based on innovative techniques:
An innovative combination of analytical separation techniques – comprehensive two dimensional liquid chromatography (2D-LC) and ion mobility (IM) - coupled with high resolution accurate mass spectrometry will be explored to obtain better qualitative and quantitative data in the context of metabolomics. The implementation of five dimensions of separation (2D-LC-IM-QTOFMS) for metabolomics will allow increased separation together with more confident identification of metabolites. This WP will include optimization of LC columns (different stationary phases), mobile phase gradients, ion mobility parameters, and mass spectrometric parameters (ionization sources, fragmentor and collision energy voltages).
WP2: Data analysis workflow
This WP is devoted to optimize data processing strategies and explore relevant bioinformatics approaches to maximally extract relevant information from the complex datasets resulting from WP1. This will need subsequent steps such as alignment of chromatograms, subtraction of the noise, and normalization of the signals. Multivariate statistical tests will then be used to detect signals that are relevant for the study set-up (e.g. differences between exposed and non-exposed cells in an exposure experiment). Identification of the signals (i.e. which metabolites) will be performed based on their retention time, CCS value, m/z value, isotope pattern and fragmentation pattern using algorithms (Molecular Formula Generator (Agilent); MetFrag/MetFusion; CFM-ID) in combination with databases (METLIN, LipidMaps, HMDB, IIMDB, MassBank) in order to putatively annotate the correct structure.
WP3: Pre-analytical phase
Apart from optimizing the instrumental analysis of (parts of) the metabolome (WP1-2), this WP will substantially improve and standardize extraction protocols in order to maximize the number of extracted metabolites in a reproducible way. Sampling and extraction protocols have to be optimized based on the matrix under investigation (e.g. cell lines, tissues, serum), but all should include critical steps such as metabolism quenching and metabolite extraction.