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, whole organism, blood, urine, etc)
Approximate mass(es) or volume(s) of samples?
Sample history
Date that samples were or are to be collected?
Preservation method?
(e.g. freeze clamping 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 extractions at Birmingham, after training by NBAF-B staff.
Total number of samples for extraction?
Preferred extraction method (e.g., Bligh-Dyer)?
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
1
H NMR spectroscopy: Non-targeted analyses that are available include 1-D NMR spectroscopy and 2-D J-resolved
NMR spectroscopy of polar metabolites (Bruker 500 or 600 MHz spectrometers).
High resolution mass spectrometry: Non-targeted analyses that are available include the measurement of polar and
nonpolar metabolites using direct infusion method (Fourier transform ion cyclotron resonance (FT-ICR) or Orbitrap
based mass spectrometers and Triversa nanoelectrospray ion source). Mass spectra can be collected in either positive or
negative ion mode, or both. In some cases, LC-MS methods will be the preferred option.
Targeted metabolite analyses will only be performed based on prior nontargeted studies.
Operation of the NMR and mass spectrometers will be conducted by NBAF-B staff only.
Number of 1-D NMR spectroscopic analyses?
Number of 2-D J-resolved NMR spectroscopic analyses?
Number of direct infusion MS analyses (positive or negative
ion mode, polar or nonpolar extracts)?
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Number of rapid UHPLC-MS analyses (either ion mode)?
Number of standard HPLC-MS analyses (either ion mode –
more sensitive, longer acquisition time)?
Spectral processing and data analysis
Raw spectral data will be processed using custom-written software (in Matlab). 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 and (for NMR) loadings / weightings plots & lists.
You will receive an initial report containing methods and results from data processing, PCA analysis, and database
searching, after which we will discuss the next steps.
It is preferable that visitors conduct their own statistical analyses at Birmingham, after training by NBAF-B staff.
What prior experience does the visitor have in multivariate
data analysis?
We will perform initial statistical analysis at NBAF-B. For further analysis, visitors will also have the
possibility to work with us in Birmingham (we offer initial training), or be sent data matrices for their own
processing.
Metabolite identification
Metabolite identification and quantification from 1-D NMR spectra can be conducted using the Chenomx NMR Suite.
Publicly available NMR libraries of pure metabolites can also be consulted; e.g., the Biological Magnetic Resonance
Data Bank and the Human Metabolome Database. In instances where confirmation of identification of a few
metabolites is crucial, biological samples can be spiked with pure metabolites and NMR spectra re-recorded.
For the FT-ICR MS data, data will initially be annotated by MS mass matching via MI-Pack 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 confirmation of identification of
a few metabolites is crucial, fragmentation experiments (MS n) can be conducted and compared to pure metabolites.
Do you anticipate requiring accurate mass and isotope
ratio measurements of particular peaks using FT-ICR MS?
(Y/N) If yes, approximately how many peaks?
Do you anticipate requiring metabolite identification via
FT-ICR MS/MS experiments? (Y/N) If yes, approximately
how many metabolites?
Do you anticipate requiring metabolite identification via
purification and further NMR and MS experiments? (Y/N) If
yes, approximately how many metabolites?
Proposed timing of visit
Preferred start date?
Estimated number of months from samples received?
If timing is critical, please explain why:
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