ARCHIVED: new version available. 2016 - Bioinformatics of METASPACE
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These slides present the bioinformatics for metabolite annotation of HR imaging MS. The bioinformatics was developed in the framework of the METASPACE project. METASPACE is a European Horizon2020 project on Bioinformatics for Spatial Metabolomics. Specifically, it aims at developing an engine for metabolite annotation of HR imaging mass spectrometry data. The project was funded in the Personalizing Health and Care program for 3 years (2015-2018) and is coordinated by the European Molecular Biology Laboratory. The presentation was given at the METASPACE Training Course at OurCon'16 on 17.10.2016. For more information on METASPACE, please visit the project website http://metaspace2020.eu, twitter @metaspace2020, or email us at contact@metaspace2020.eu.
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ARCHIVED: new version available. 2016 - Bioinformatics of METASPACE
- 1. METASPACE TRAINING COURSE PART 1. THEORY Metabolite annotation In HR imaging MS Theodore alexandrov EMBL / UCSD / SCILS @thalexandrov
- 2. m/z 203.103 m/z 212.852 m/z 223.075 C. albicans E. coli with Alex Koumoutsi, Nassos Typas @ EMBL C. albicans E. coli metabolite identification
- 3. dataset 100 GB 100.000 spectra 10.000.000 images metabolome 50.000-100.000 molecular structures Metabolite annotation Of 10.000.000 ion images in-source fragmentation ion adducts m/z A m/z B m/z C AMP MW 347.063 [M+H]=348.071 For 50K moleculaR structures Dark matter isotopologues
- 4. Targeted metabolite imaging howto 1. Consider possible adducts – +H, +Na, +K or –H, –Cl 2. Calculate m/z of each adduct – principal or monoisotopic 3. Examine ion images 4. Examine the potential isotopic pattern 5. Estimate the ambiguity (any isomers? isobars?) 6. Validate with in situ MS/MS – On a region of high intensity
- 6. Palmer et al., Nature Methods, accepted Molecular annotation Fdr calculation
- 7. Molecular annotation Fdr calculation 1. Consider possible adducts 2. Calculate m/z of each adduct 3. Examine images 4. Examine the potential isotopic pattern 5. Estimate the ambiguity (any isomers? isobars?) 1 3 4 3 2 5
- 8. Measure of Spatial chaos Structured informative Chaotic non-informative
- 9. Spectral & spatial isotope scores 9 monoisotopic image structured? ok fine isotope structure matching theor? ok isotopic images co-localized? not è doesn‘t pass the filters
- 10. Molecular annotation Fdr calculation Ok, all ions are scored by their likelihood ... But how to choose the cutoff?
- 11. How to choose msm cutoff
- 12. how to select parameters in proteomics Database Data Molecular Iden=fica=on List of molecules Correct? 1. How to quantify correctness? 2. False Discovery Rate FDR = ratio of false positives 3. Don’t know false positives è cannot calculate FDR 4. Can we estimate it? true positives false positives
- 13. How to estimate fdr In proteomics Database Data Molecular Iden=fica=on Molecular IDs Fake database Molecular IDs “Decoy” “Target” FDR # false positives for target # identifications for target estimated FDR # target FPs # target IDs = ≈ # decoy FPs # target IDs true positives false positives false positives positives: = = Target similar to Decoy defini3on =
- 15. you won tickets to chile!
- 16. which cities are sunny? Whom to believe? 1. Let’s take cities which we know answers for (always rainy) = decoy 2. Ask to predict what the weather will be like there “sunny” they say è false positive (# decoy FPs)
- 17. FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR DR DR DR DR DR DR DRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR HM... How is it related to imaging ms?! FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDR FDRFDRFDR FDRFDR FDR FDR FDR FDR FDR FDR FDRFDR FDRF
- 18. FDR for imaging ms Fdr calculation Palmer et al., Nature Methods, accepted
- 19. Fdr-controlled metabolite annotation Palmer et al., Nature Methods, accepted
- 20. Does it really work?
- 21. Test case mouse brain sections Reference H&E images from Allen Brain Atlas Bregma 1.42mm 3 serial sections Bregma -1.46mm 1 section Bregma -3.88mm 1 section animal 1 animal 2
- 22. Imaging ms DHB matrix ImagePrep 50 μm pixel size 10K spectra 100-1.200 m/z 20-30 GB ► ► solarix XR 7T Paracell 130K @ m/z 400 with Regis Lavigne, Charles Pineau @ UR1, France
- 28. @ EMBL Andy Palmer, Prasad Phapale Vitaly Kovalev, Sergey Nikolenko Artem Tarasov, Dominik Fay Luca Rappez Thank you METASPACE Consortium Christoph Steinbeck, EMBL-EBI Lennart Martens, VIB Charles Pineau, Regis Lavigne, URennes Pieter Dorrestein, UCSD Zoltan Takats, Kirill Veselkov, ICL Dennis Trede, SCiLS Oliver Panzer, ERS and their team members Bruker Michæl Becker, Jens Fuchser @alexandrovteam European Horizon2020 HEALTH programme