Canadian Bioinformatics Workshops

1. Canadian Bioinformatics Workshops www.bioinformatics.ca

2. Module #: Title of Module 2

3. Module 2 Software for Metabolite Identification and Quantification David Wishart Informatics and Statistics for Metabolomics May 3-4, 2012

4. 25 PC2 20 15 ANIT 10 5 0 -5 Control -10 -15 PAP -20 PC1 -25 -30 -20 -10 0 10 ppm 7 6 5 4 3 2 1 TMAO creatinine hippurate allantoin creatinine taurine citrate urea hippurate 2-oxoglutarate water succinate fumarate ppm 7 6 5 4 3 2 1 2 Routes to Metabolomics Quantitative (Targeted) Methods Chemometric (Profiling) Methods

5. The Problem With Metabolomics Gene IDs + Transcript Abundance Genomics Protein IDs + Concentrations Proteomics ? Metabolite IDs + Concentrations Metabolomics

6. Metabolite Identification • Two scenarios identification of “known unknowns” and “unknown unknowns” • For “known unknowns” use spectral or metabolite libraries to ID and quantify via spectral deconvolution • For “unknown unknowns” (truly novel) use computer-aided structure elucidation methods (CASE) “…there are known unknowns; that is to say we know there are some things we do not know. But there are also unknown unknowns -- the ones we don't know we don't know.”

7. Spectral Deconvolution • Specifically for “Known Unknowns” • Matches peaks to a known set of peaks (from pure cmpds) in a pre-compiled database • Can be done with NMR, GC-MS, LC-MS and MS/MS data • Called “Targeted” or quantitative profiling

8. Metabolite ID by Spectral Deconvolution (NMR) Mixture Compound A Compound B Compound C

9. Chenomx & AMIX www.bruker-biospin.com www.chenomx.com

10. Why Chenomx? • Canadian software company • Compatible with most NMR formats • Simple-to-use software for 1D NMR • Large library of 450 reference spectra

11. Download Software/Demo

12. Chenomx NMR Suite • Chenomx Processor - Spectral Phasing - Water Deletion/Suppression - Baseline Correction - Referencing and Ref. Deconvolution • Chenomx Profiler - Compound Library (Reference Spectra) - Supports Spectral Profiling/Deconvolution

13. Processor Steps • Launch Processor program • Select and upload spectrum (File Open) • Confirm spectral parameters • Phase spectrum (Under Processing) • Find DSS reference • Remove water peak (Under Processing Advanced) • Perform baseline correction (Under Processing) • Perform reference deconvolution (Under Processing Advanced)

14. Select/Upload Spectrum

15. Set/Confirm Parameters

16. Raw Spectrum Zoomed View

17. NMR Spectra Need “Fixin’” Before After Baseline correction Water suppression Referencing Shimming Phasing

18. Under Processing Menu – Choose Your Options

19. Phase Spectrum After auto phasing, do manual phasing as necessary

20. NMR Phasing

21. Find DSS Peak Automatically

22. Remove Water Peak After Before

23. Baseline Correction

24. Baseline Correction – Auto Spline Zoomed View

25. Reference Deconvolution - 1

26. Reference Deconvolution - 2

27. Spectrum after Processing

28. Profiler Steps • Launch Profiler program • Select and upload spectrum (File Open) • Select “Profiled Compounds” and “500 MHz” • Always begin by profiling DSS (there are 4 major DSS peak clusters) • Try to fit/drag until the green subtraction line is essentially flat • Once finished with DSS, select other compounds on the compound list and proceed to “tweak” their fits • When finished, export the concentration list

29. Launch Chenomx Profiler

30. Select Appropriate Spectral Library

31. Profile DSS

32. Profile DSS (Click on 0.0 Cluster and Fit)

33. Profile DSS (Click on Other Clusters)

34. Click and Drag Peak Shapes and Positions

35. Example 1: Fit Acetate (compound with single peak)

36. Example 2: Fit Alanine

37. Data Export

38. Now It’s Your Turn…http://bioinformatics.ca/workshop_wiki/