addressing a key challenge in non-targeted environmental...
TRANSCRIPT
Addressing a key challenge in non-targeted environmental metabolomics: identifying the
parts list through Deep Metabolome Annotation
Mark Viant, University of Birmingham, UK
SETAC/iEOS Omics Meeting, Ghent, Belgium 13th September 2016
1. Genomes but no metabolomes!?
2. Metabolomics workflows and metabolite identification
3. Where next? - Deep Metabolome Annotation
Overview
Human Genome Project
•8.7 mio eukaryotic species on earth (±1.3 mio) • 1.2 mio species identified and classified
•3000 - 4000 complete species genomes sequenced
What about completed metabolomes?
Triggered explosion of activity…
Christoph Steinbeck, EBI
Metabolomics (publishing) has taken off worldwide
Christoph Steinbeck, EBI
Metabolomics (data) is rapidly increasing (at EBI)
Christoph Steinbeck, EBI
Yet near-zero activity on experimentally characterising metabolomes!!
1. Where are the metabolomes?
2. Metabolomics workflows and metabolite identification
3. Where next? - Deep Metabolome Annotation
Overview
Biological study e.g. exposure expt
Measure metabolites (peaks) e.g. mass spectrometry, NMR
Data processing & statistical analysis (of peaks)
Biological interpretation
Experimental design
Generic metabolomics workflow
(Attempted) identification of limited number of ‘interesting’ peaks
1. Database searches 2. Compare to pure
standards 3. De novo identification
(Natural Products Chem) 4. ???
Daphnids A
Nanosun™ ZnO nanoparticles
Example 1 – identification of “interesting” peaks
Statistical analyses revealed multiple (correlated) peaks that decreased upon ZnO
NP exposure
Extract samples: organic solvents Mass spectrometry: MS/MS, MSn fragmentation
Example 1 – identification of “interesting” peaks
1E20N_neg_277-137_CID #1-190 RT: 0.00-1.03 AV: 190 NL: 7.36T: ITMS - p ESI Full ms3 [email protected] [email protected] [50.00-300.00]
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81.08 109.0865.00 73.17 93.0083.08 97.08 101.42
1E20N_neg_277-165_CID #1-185 RT: 0.00-1.01 AV: 185 NL: 8.51T: ITMS - p ESI Full ms3 [email protected] [email protected] [50.00-300.00]
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85.0880.00 137.0073.00 121.08 164.33 165.7599.2564.92 95.83 107.17 146.7557.17 124.92
1E20N_neg_277-165_CID_100826160620 #1-185 RT: 0.00-1.01 AV: 185 NL: 4.68T: ITMS - p ESI Full ms3 [email protected] [email protected] [50.00-300.00]
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85.08 137.0073.00 164.00107.00 121.0857.17 62.67 93.17 142.08125.17
1E20N_neg_277_CID #1-225 RT: 0.00-1.00 AV: 225 NL: 1.48E3T: ITMS - p ESI Full ms2 [email protected] [75.00-300.00]
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165.08209.1799.0080.00 167.08 249.17233.25 259.17164.08147.00129.17 197.17110.00 294.92
x5 x5
28 68 110 138 180
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Revealed that sulfated lipids decreased concentration upon exposure to the nanoparticle
No pure standards available – a common problem!
Marine bacteria (Rhodobacteraceae) can use sulfur compounds as
supplementary energy sources - Effect of thiosulfate supplementation?
PCA loadings plot
Peaks of ‘interest’ that changed with thiosulfate supplementation were not in any databases!
Example 2 – identification of “interesting” peaks
B-
O
O
O
O
O
O
O
O
Boron containing metabolites are known to have roles in cell-to-cell communication, communication among bacteria, antibiotic properties (Dembitsky et al., Chem. Rev., 2011, 209-237)
This approach is not scalable!
Extract samples: SAX, SCX, HILIC SPE NMR: Structural elucidation (1H NMR, HSQC, HMBC)
Mass spectrometry: MS/MS, MSn fragmentation
Example 2 – identification of “interesting” peaks
• Biodiversity: 1000’s of species, 1000’s of metabolomes • Microbiomes too!
Endogenous metabolites (>10,000 forming endometabolome)
Xenobiotics (>>1,000? - exposome)
Uptake, metabolism & effect of
xenobiotics on organism health
Chemical signalling (exometabolome)
Complexity of what we are trying to measure in environmental metabolomics
Metabolite identification –
A BOTTLENECK IN METABOLOMICS
A For metabolomics to be successful it is essential to derive biological
knowledge from analytical data - a view emphasised by a Metabolomics ASMS Workshop Survey 2009 which found that the biggest bottlenecks in metabolomics were thought to be identification of metabolites (35%)
and assignment of biological interest (22%)
http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics-Survey-2009
1. Where are the metabolomes?
2. Metabolomics workflows and metabolite identification
3. Where next? - Deep Metabolome Annotation
Overview
Comprehensive database of 1000’s of identified metabolites for each
Model Organism Metabolome (open access) International coordination:
Metabolomics Society MOM Task Group
Existing expt’al observations from
literature (text mining) Predicted metabolism: genome wide metabolic
reconstruction
New expt’al data: more exhaustive
analytical methods
Focus on Model Organism Metabolomes
‘Deep Metabolome Annotation’ (DMA) 2012-present
• Multi-platform analytical characterisation: extensive extraction & fractionation chemistries, chromatography (LC, GC,...), detectors (mass spectrometry, NMR spectroscopy...)
• Databases: new local database, mzCloud and MetaboLights
• Part of University of Birmingham’s Technology Alliance Partnership with Thermo Fisher Scientific
DMA of Daphnia magna
• Keystone species of freshwater ecosystems • Eco-toxicological model organism (OECD) • NIH model organism for biomedical research • Studied at all functional levels by international community
To-date, fewer than 200 metabolites reported in literature
DMA status
• ca. 80% of workflow development completed • ca. 20% of LC-MS and DIMSn (Orbitrap) data recorded • All NMR and GC-Orbitrap data recorded
Take-home messages
• Current capability to annotate & identify metabolites is
unacceptable, strongly inhibiting the field of
metabolomics
• Activity is building, globally, to drive forward the
characterisation of Model Organism Metabolomes as a
key starting point
• Experiments are underway to experimentally discover
the first comprehensive metabolome library of a model
organism – Daphnia magna
• Mr Martin Jones • Mr Thomas Lawson • Dr Ralf Weber • Dr Warwick Dunn
• Dr Tony Edge (Hemel Hempstead, UK) • Dr Ken Cook (Runcorn, UK) • Dr Alex Adam (Hemel Hempstead, UK) • Dr Tim Stratton (San Jose, CA, USA) • Many others…
Acknowledgements