1. Canadian Bioinformatics Workshops www.bioinformatics.ca

2. 2Module #: Title of Module

3. Module 2 Software for Metabolite Identification and Quantification

4. Learning Objectives To learn about the concepts of spectral deconvolution for metabolite identification and quantification To learn how to use spectral deconvolution for NMR-based metabolomics (Chenomx) To try and “deconvolve” a real biofluid spectrum using Chenomx software

5. 2 Routes to Metabolomics 1234567ppm Quantitative (Targeted) Methods Chemometric (Profiling) Methods

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

7. 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.”

8. 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

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

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

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

12. Download Software/Demo

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

14. Processor Overview Sidebar View Processing History Processor Dashboard Status Bar Thumbnail Spectral View

15. 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 Region Deletion) Perform baseline correction (Under Processing) Perform reference deconvolution (Under Processing Shim Correction)

16. Select/Upload Spectrum

17. Set/Confirm Parameters

18. Raw Spectrum Zoomed View

19. NMR Spectra Need “Fixin’” Before After Referencing Baseline correction Region Deletion Phasing Shimming

20. Under Processing Menu – Choose Your Options

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

22. NMR Phasing

23. Remove Water Peak Before After

24. Baseline Correction

25. Baseline Correction – Auto Spline Zoomed View

26. Reference Deconvolution - 1

27. Reference Deconvolution - 2

28. Spectrum after Processing

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

30. Profiler Overview Sidebar View Legend Cluster Navigator Cluster Navigator Compound Table Spectrum View Quick Search Thumbnail Status Bar

31. 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

32. Launch Chenomx Profiler

33. Select Appropriate Spectral Library

34. Profile DSS

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

36. Profile DSS (Click on Other Clusters)

37. Click and Drag Peak Shapes and Positions

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

39. Example 2: Fit Alanine (Auto Fit)

40. Example 2: Fit Alanine

41. Data Export

42. Now It’s Your Turn… http://bioinformatics.ca/wor kshop_wiki/