Phenome Centre Birmingham offers a complete collaborative service to provide expertise and advice to perform metabolic phenotyping studies from conception and experimental design through data acquisition to data analysis and biological interpretation.
Sample types: We provide the capabilities to study a diverse range of sample types. For large-scale metabolic phenotyping studies we recommend the use of biofluids including serum, plasma and urine. We also provide the capability to analyse other biofluids (e.g. CSF), tissues (e.g. liver or muscle) and cell lines.
Non-Targeted Metabolomics: applied when a broad survey of different classes of metabolites is required in discovery-based studies and provides relative quantification data on thousands of metabolites. We have an established analytical workflow that combines state of the art nuclear magnetic resonance (NMR) spectroscopy and ultra-high performance chromatography coupled with high resolution mass spectrometry (UHPLC-MS) to produce high quality data representing thousands of metabolites present in diverse areas of metabolism including amino acid metabolism, lipid metabolism and purine metabolism.
Four specific analytical methodologies are applied:
- 1H NMR spectroscopy for a broad coverage of high concentration lipophilic and hydrophilic metabolites; typically up to 100 metabolites are detected and quantified
- Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC-MS) for a broad coverage of hydrophilic metabolites
- Reversed phase UHPLC-MS for broad coverage of lipophilic metabolites including for lipidomic studies.
- Biocrates p400 kit applying Direct Infusion Mass Spectrometry and UPLC-MS
To ensure the quality of data meets defined acceptance criteria our fully-trained team applies standard operating procedures and quality control samples for all studies. The data is processed, interrogated and analysed using standardised computational workflows. Our workflows include tools to visualise data quality, to apply biostatistics, and to identify metabolites. The identification of biologically or clinically significant metabolic markers (biomarkers), derived from biostatistical analysis and initial metabolite annotation, can be validated by the use of targeted assays (see below - Targeted Metabolomics).
Our facilities include:
- Four Acquity UPLC systems with 2777c sample managers coupled to electrospray Xevo G2-XS QTof mass spectrometers (Waters)
- Three Ultimate3000 RSLC UHPLC systems coupled to electrospray Q Exactive Focus mass spectrometers (Thermo Scientific)
- Two 600MHz NMR spectrometers each with a room temperature 5mm probe (Bruker)
Targeted Metabolomics: applied when a small and pre-defined set of metabolites are required to be investigated and provides absolute quantification data on tens of metabolites. In cases where the metabolites of interest are known we offer targeted assays to detect and quantify a small number of related metabolites. These metabolites may be linked based on metabolic class or biological function (e.g. amino acids) to allow a specific metabolic pathway to be studied or can be metabolic biomarkers from different metabolic pathways requiring validation and subsequent translation in to clinical use. Metabolites are quantified using targeted analytical methods on triple quadrupole mass spectrometers coupled with ultra-high performance chromatography, which produces true quantitative data. A range of targeted assays are available and research-specific targeted assays can be developed on request.
Our facilities include:
- Two Ultimate3000 RSLC UHPLC systems coupled to electrospray Quantiva triple quadrupole mass spectrometers (Thermo Scientific)
- One Acquity UPLC system with 2777c sample manager coupled to electrospray Xevo TQ-S triple quadrupole mass spectrometer (Waters)
Compound Identification: Often within the scientific literature the statistically significant and biologically important metabolites discovered in non-targeted metabolomics studies are not identified. Biological interpretation and validation of biomarkers require the chemical identification of the metabolites discovered in non-targeted studies. If sufficient sample is available then metabolite identification can be performed in Phenome Centre Birmingham by a combination of solvent/SPE/chromatographic fractionation, NMR spectroscopy and sequential mass spectrometry fragmentation experiments (MSn).
Our facilities include one electrospray LTQ-Orbitrap Elite mass spectrometer equipped with nanoLC and 2D-LC capabilities (Thermo Scientific) and nanoelectrospray infusion capabilities using the Advion Triversa Nanomate system
Data Processing, Biostatistical Analysis and Metabolite Annotation: The data is processed, interrogated and analysed using standardised and reproducible computational workflows. The workflows include tools, written in the R programming language and Python, to visualise data quality, to apply biostatistics, and to annotate metabolites or potential biomarkers. The workflows have been integrated into the Galaxy workflow management system [https://galaxyproject.org]. We apply a range of univariate and multivariate statistical approaches, dependent on the experimental design, to robustly discover metabolites that contribute to the differences related to the biological or clinical question.