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Transcript of NMR Automation
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NMR Automation: Bayesil, NMRlib and beyond
Philip LiuApril 11, 2013
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NMR Automation Overview• 1. NMR a) history b) Automation• 2. ChenoMX
a) Processorb) Profilerc) Compound Builder
• 3. XML• 4. NMRlib
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1. NMR History• 1945 - First
experimentally observed by Felix Bloch (Stanford) and Edward Purcell (Harvard)
• 1946 - Spectra first published in January edition of Physical Review
• 1952 - Nobel prize for physics shared by Bloch and Purcell
Felix Bloch
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NMR (Nuclear Magnetic Resonance)
• Nuclei of atomic isotopes (1H, 13C, 31P, 15N, 29Si etc.) interact with a static magnetic field
• Magnetic field makes spin states of nuclei differ in energy, and transitions between energy states can be observed
• Chemically distinct nuclei differ in resonance frequency in the same magnetic field –
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NMR• Nuclei are aligned by a magnetic field • An oscillating, perpendicular magnetic field is
applied
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NMR• Precession is a change in the
orientation of the rotational axis of a rotating body.
• Dependent on rate of spin, size and shape of gyro, strength of gravity.
• Find out the magnetic field strength to get the precession frequency (frequency it takes to get the nucleus precessing)
• 2nd magnetic field varies in time at the same rate as the precession - i.e. resonant with the nuclear motion (continuous wave NMR)
• Nuclei which are not aligned
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• This non-equilibrium magnetisation can be induced, generally by applying a pulse of resonant radio-frequency close to the Larmor frequency of the nuclear spins.
• Fourier Transform NMR, free induction decay (FID) is the observable NMR signal generated by non-equilibrium nuclear spin magnetisation precessing about the magnetic field.
• The resultant oscillating magnetization vector of the nuclei induces a current in a pickup coil, creating an electrical signal oscillating at the NMR frequency (aka FID).
• The FID contains the vector sum of the NMR responses from all the excited spins
• To obtain the frequency-domain NMR spectrum (NMR absorption intensity vs. NMR frequency) this time-
NMR - FID
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NMR Spectra• The chemical shift of a nucleus is the
difference between the resonance frequency of the nucleus and a standard, relative to the standard. (We use DSS: 4,4-dimethyl-4-silapentane-1-sulfonic acid)
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NMR Automation• Why Automation?-time: countless hours
spent processing/profiling spectra
-inconsistencies between different users (user bias)
-misidentification in profiling
-user fatigue results in profiling errors
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NMR AutomationAutomation-time: Faster (minutes vs.
hours)-consistent profiling-misidentification also
consistent, can correct and re-run spectra (saves time – hopefully!)
-fatigue is negligible
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NMR Automation Workflow
ChenoMX: Processor
NMRlib
ChenoMX: Profiler
NMR
XML
Automation Standards (HMDB)
Samples
BAYESIL (Automation
Program)
.fids (raw ChenoMX: Compound
Builder
.fids (raw
Astounding results
Library creation
Sample processing
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2. ChenoMX
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ChenoMX Processor Overview (Pre-processing)
• 1. Open Processor, Open .fid file• 2. Phase • 3. Calibrate CSI• 4. Region Deletion (water removal)• 5. Baseline Correction– 6. Line Broadening (optional)
• 7. Shim Correction (optional)• 8. Calibrate CSI• Send to ChenoMX Profiler
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ChenoMX Processor• Pre-processing of raw NMR files (.fid)• .cnx files
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Open Processor
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1. Open Spectra (.fid) - Ethanol
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Phasing• Phasing corrects shifts that may have occurred
in data acquisition• Asymmetric peaks/clusters may be inverted• Larger phase shifts may have an oscillation to
the baseline• 3 phase angle controls: normal (1°), fine (0.1°)
and very fine (0.01°)
Sorry, William Shatner, not this kind of
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2. Phase
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First order phase
Zero order phase
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Auto – Phase (sometimes works)
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Make peaks symmetric by adjusting the sliders below (manual phasing), accept
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3. Calibrate CSI (Chemical Shift/Shape Indicator)
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Calibrate automatically, check DSS concentration, accept
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4. Region Deletion (Water removal in versions older than 7.0)
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Example: GlucoseCheck for peaks you may be deleting!!
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You can click and drag the blue lines/region or type in values.No peaks inside blue area, accept!
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5. Baseline CorrectionRemoves distortion in
spectrum Allows for accurate
quantification
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Baseline Correction – auto linear
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Baseline Correction – auto spline
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• Move blue dots to centre of noise,
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If you see wild distortions, do not panic. Move points to centre of noise and it will smooth out – hold control and click on the line to add points, hold shift and click to
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Line Broadening (Optional)• Multiplies fid by an exponential
function before fid is Fourier transformed
• Increases linewidth in spectrum and smoothes out instrument noise
• Broader peaks, less noisy• Without broadening – sharper peaks
but baseline may be noisy
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OPTIONAL6. Line
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Line broadening: adjust Hz in
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7. Shim Correction
Creates an ideal spectrum, removes lineshape distortions based
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Shim correction -
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8. Calibrate CSI
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Calibrate CSI (Calibrate Automatically)
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Congratulations, Processing completed, send to Profiler!
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ChenoMX Profiler• Compound identification• .cnx file
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Type compound name here (start with DSS), make sure correct library is selected
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Black: SpectrumRed: Sum of profiled compoundsGreen: Subtraction lineBlue: Current compound selectedShortcuts: Space – automatic fit(bottom left will say it can’t fit if autofit doesn’t work)
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These numbers will go green if there’s a good fit (NB: will not go green all the time even if fit well)Click these numbers to view/move different clustersNB: Name here is the compound selected
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Bottom blue arrow will shift cluster selected on x axisRight side arrow will shift all clusters on y axis Clicking and dragging on the blue peak will move all clusters x + y
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Fit clusters, note concentration of metabolite/DSS
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Compound Builder• Creates library files for ChenoMX• .xcpd files
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Open Compound Builder
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Overlay .cnx file (ChenoMX file created by processor/profiler)
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Open .cnx
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Edit Compound Details: name, author, magnet frequency and concentration
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Ethanol
DSS
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Right click peak, add new peak to new cluster
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Right click, add new peak to existing cluster
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NB: Blue denotes peaks currently selected
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Automatic fit, you can press this multiple times.
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Message that program cannot fit any longer, ok.
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Adjusting peaks manually, - matching blue peaks/red line (sum line) to match the black lines (actual spectra)
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Can select and group clusters (Control + G) or right click
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Success! Save.
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3. XML • Extensible Markup
Language (.xml files)• A (document) markup
language is a modern system for annotating a document in a way that is syntactically distinguishable from the text. [Wikipedia]
• Advantage: Machine and Human readable
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Conversion from .xcpd XML file
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XML• File Header (red – edit these fields)• <name>Ethanol</name>• <ID>HMDB00108</ID>• <resonatingElement>hydrogen</
resonatingElement>• <libraryVersion>experimental</
libraryVersion>• <metabolicGroup>all</metabolicGroup>• <type>hnmrv</type>• <magnetFreq>500</magnetFreq>• <quantification>1.2863</quantification>*
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More XML
• <clusters>• <cluster>• <center>1.17166</center>• <lowerBound>1.17166</
lowerBound>• <upperBound>1.17166</
upperBound>• <peaks>• <peak>• <center>1.15747</center>
Bounds limit how far the cluster centres can shift
Peaks are described by their centres,
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BAYESIL • “Siamak’s Program”• Processes NMR spectra similarly to
ChenoMX – phases, baseline corrects etc.
• Global fit – uses area under the curve to fit spectra
• Future improvements: UI (User interface), flagging false positives/negatives, concentration corrections,
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BAYESIL
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Automated
Human
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4. NMRlib• “Roman’s Program”• GUI (Graphical User Interface), web
based application (nmrlib.wishartlab.com) that uses BAYESIL to process spectra and create XML files
• Faster than ChenoMX approach, eliminates typographical errors
• Doesn’t allow for overlapping peaks
NMRlib
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NMRlib Search, sort
files by user, date, name, magnet frequency,
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NMRlib• Fill in fields, Name,
HMDBID, NMR Freq. Metabolite concentration (as measured by ChenoMX or experimental), and ratio of [metabolite]/[DSS]
• -Spectrum files must be .zip files and the fid must be in a folder named HMDBxxxxx.fid eg.
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NMRlibPick peaks
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Min. peak height: deletes peaks below __
Noise factor: higher # = shows less points
lower # = shows more
Peaks picked by program, DSS is
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• Peaklist: delete peaks
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Adding peaks (click spectra)
1
2
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Peak added!
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Name clusters, save
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Alternative method of creating clusters: input range (ppm)
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Confirm clusters
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Run fitting!
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Success? Not so fast! Examine fit
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1. Check fit
2. Approval 3. back
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Right click HMDB0008.xml, save link as
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Save, SUCCESS!
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Future • Finish making xmls for all these
compounds • Profile Serum (~40 cmpds) and Urine
(200+ compounds)
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Thank you! (You can wake up now)
• Dr. Wishart• Igor, Trent, Rupa, Edison• Siamak, Roman• Wishartlab
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Questions?
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References• http://chemnmr.colorado.edu/moreinfo/whatisnmr.html• http://www.google.ca/imgres?imgurl=http%3A%2F%2Fwww.riken.jp%2Fengn%2Fr-
world%2Finfo%2Frelease%2Fnews%2F2003%2Faug%2Fimage%2Fefg-2.jpg&imgrefurl=http%3A%2F%2Fwww.riken.jp%2Fengn%2Fr-world%2Finfo%2Frelease%2Fnews%2F2003%2Faug%2Findex.html&docid=IDBu8C4qtKDVSM&tbnid=npBbvuV7tHmVLM&w=246&h=146&ei=mCZnUc2DB6ayigKl04GwDQ&ved=0CAUQxiAwAw&iact=ricl
• http://edwebproject.org/india/pics/swing.jpg• http://www.abc.net.au/science/articles/2011/03/29/3175747.htm• http://www.planetseed.com/node/15291• http://en.wikipedia.org/wiki/File:Earth_precession.svg• http://www.physiopro.co.za/the-head-work-behind-your-headache/#• http://4.bp.blogspot.com/-roZe_7I5lJ4/TWxGXMuRoDI/AAAAAAAABSE/
fe5qFP_iGBw/s1600/BERTI_robot.jpg• http://i.current.com/images/asset/900/821/06/4Fo01x.jpg• http://i.telegraph.co.uk/multimedia/archive/00802/460-basil_802346c.jpg• http://www.chenomx.com/support/img/page58/Chenomx%20NMR%20Suite
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