Canadian Bioinformatics
Workshops
www.bioinformatics.ca
Module #: Title of Module
2
Module 2
Software for Metabolite Identification
and Quantification
David Wishart
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
2 Routes to Metabolomics
ppm
7
6
5
4
Quantitative (Targeted)
Methods
3
2
Chemometric (Profiling)
Methods
25
TMAO
hippurate
allantoin creatinine taurine
1
PC2
20
creatinine
15
10
citrate
ANIT
5
hippurate
urea
2-oxoglutarate
water
succinate
fumarate
0
-5
-10
ppm
7
6
5
4
3
2
1
Control
-15
PAP
-20
-25
-30
PC1
-20
-10
0
10
The Problem With Metabolomics
Gene IDs +
Transcript
Abundance
Genomics
Protein IDs +
Concentrations
Proteomics
?
Metabolomics
Metabolite IDs +
Concentrations
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.”
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
Metabolite ID by Spectral
Deconvolution (NMR)
Mixture
Compound A
Compound B
Compound C
Chenomx & AMIX
www.bruker-biospin.com
www.chenomx.com
Why Chenomx?
•
•
•
•
Canadian software company
Compatible with most NMR formats
Simple-to-use software for 1D NMR
Large library of 450 reference spectra
Download Software/Demo
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
Processor Overview
Sidebar View
Processing History
Processor Dashboard
Spectral View
Thumbnail
Status Bar
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)
Select/Upload Spectrum
Set/Confirm Parameters
Raw Spectrum
Zoomed
View
NMR Spectra Need “Fixin’”
Before
After
Baseline
correction
Shimming
Region
Deletion
Referencing
Phasing
Under Processing Menu –
Choose Your Options
Phase Spectrum
After auto phasing, do manual phasing as necessary
NMR Phasing
Remove Water Peak
Before
After
Baseline Correction
Baseline Correction –
Auto Spline
Zoomed
View
Reference Deconvolution - 1
Reference Deconvolution - 2
Spectrum after Processing
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
Profiler Overview
Sidebar View
Legend
Cluster
Navigator
Spectrum View
Quick Search
Thumbnail
Compound Table
Status Bar
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
Launch Chenomx Profiler
Select Appropriate Spectral
Library
Profile DSS
Profile DSS (Click on 0.0
Cluster and Fit)
Profile DSS (Click on Other
Clusters)
Click and Drag Peak Shapes
and Positions
Example 1: Fit Acetate
(compound with single peak)
Example 2: Fit Alanine (Auto Fit)
Example 2: Fit Alanine
Data Export
Now It’s Your Turn…
http://bioinformatics.ca/wor
kshop_wiki/