Appendix. Services offered by the NERC Biomolecular Analysis Facility - Birmingham, NBAF-B

Metabolomics Analysis

Please include a short description of the overall experimental design on the main application form.

Sample description
Full name and taxonomic classification of study species?
Type(s) of samples for analysis?(e.g. type of tissue)
Approximate mass(es) or volume(s) of samples?
Sample history
Date that samples were or are to be collected?
Preservation method?(e.g. freezing in liquid nitrogen)
Storage method and storage temperature since collection?
Metabolite extraction from biological matrices
Polar and non-polar metabolites will usually be isolated by liquid-liquid extraction (e.g. methanol/chloroform/water).It is preferable that visitors conduct their own metabolite extractionsat Birmingham, after training by NBAF-B staff.
Total number of samples for extraction?
Preferred extraction method (e.g., methanol/chloroform/water)?
What prior experience does the visitor have in metabolite extractions?
In what capacity will NBAF-B staff be required?
(e.g. for initial training only)
Metabolite analysis
Non-targeted1H NMR spectroscopy: analyses that are available include 1-D NMR spectroscopy and 2-D J-resolved NMR spectroscopy of polar metabolites (Bruker 600 MHz spectrometers). One or both methods are applied dependent on the amount of sample available.
Non-targetedmass spectrometry (MS): analyses that are available include the measurement of polar and nonpolar metabolites using nanoelectrospray direct infusion MS (DIMS) and/or liquid chromatography MS (LC-MS). Mass spectra are collected in positive and/or negative ion modes.Choice of method depends on amount of sample available.
Operation of the NMR and mass spectrometers will be conducted by NBAF-B staff only.
Number of 1-D NMR / spectroscopic analyses of polar extracts?
Number of 2-D J-resolved NMR spectroscopic analyses of polar extracts?
Number of direct infusion MS analyses (clarify positive and/or negative ion mode, polar and/or nonpolar extracts)?
Number of LC-MS analyses (clarify positive and/or negative ion mode, polar and/or nonpolar extracts)?
Spectral processing and data analysis
Raw spectral data will be processed by NBAF-B staff using custom-written software. Subsequent data analysis will comprise of univariate and/or multivariate methods,e.g. principal components analysis (PCA), partial least squares regression (PLS-R), or PLS discriminant analysis (PLS-DA), mostly based on the Eigenvector PLS Toolbox.
Format of NMR data from facility: matrix containing samples (rows) x NMR bins/buckets (columns).
Format of MS data from facility: matrix containing samples (rows) x MS peaks (columns).
Format of output from univariate analyses: individual NMR bins/buckets or MS peaks, and their associated p or q values and fold-changes.
Format of output from multivariate analyses: scores plots and loadings / weightings plots (with importance of each peak reported from models).
You will receive an initial report containing methods and results from data processing, PCA analysis, and database searching, after which we will discuss whether further statistical analysis is required. NBAF-B is only funded to undertake a core level of statistical analysis; more advanced analyses could be undertaken by the visitor at their home institution.
It is preferable that visitors conduct their own statistical analysesat Birmingham, after training by NBAF-B staff.
What prior experience does the visitor have in univariate and multivariate data analysis?
Metabolite annotation
Metabolite annotation and quantification from 1-D NMR spectra can be conducted using the Chenomx NMR Suite. Publicly available NMR libraries of pure metabolites canalso be consulted; e.g., the Biological Magnetic Resonance Data Bank and the Human Metabolome Database. Metabolite annotation is conducted to the Metabolomics Standards Initiative level 2 (2007). In instances where identification of a few metabolites is required, NMR spectra of pure standards (supplied by the visitor) can be recorded, including spiking.
For mass spectra, data will initially be annotated by MS mass matching via MI-Pack and/or PUTMEDID using public metabolite databases; e.g. KEGG. Empirical formulae (CcHhNnOoPpSs) can be annotated with higher accuracy for each peak using higher accurate mass and approximate isotope ratio measurements.In instances where identification of a few metabolites is required, fragmentation experiments (MSn) of pure standards (supplied by the visitor) can be attempted and compared to MSn of the biological samples.
Do you anticipate requiring metabolite identification via NMR experiments? (Y/N) If yes, approximately how many metabolites?
Do you anticipate requiring metabolite identification via MSnexperiments? (Y/N) If yes, approximately how many metabolites?
Proposed timing of visit
Preferred start date?
Acceptable number of monthsfor project, from receipt of samples at Birmingham?
If timing is critical, please explain why:

Note: the Birmingham Metabolomics Training Centre( offers a range of introductory and advanced courses, both face to face and online, that may benefit applicants either prior to or during a project.

NBAF-B Appendix Version 11.09.2017