NMR Automation: Bayesil, NMRlib and beyond

Philip LiuApril 11, 2013

NMR Automation Overview• 1. NMR a) history b) Automation• 2. ChenoMX

a) Processorb) Profilerc) Compound Builder

• 3. XML• 4. NMRlib

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

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 –

NMR• Nuclei are aligned by a magnetic field • An oscillating, perpendicular magnetic field is

applied

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

• 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

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)

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

NMR AutomationAutomation-time: Faster (minutes vs.

hours)-consistent profiling-misidentification also

consistent, can correct and re-run spectra (saves time – hopefully!)

-fatigue is negligible

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

2. ChenoMX

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

ChenoMX Processor• Pre-processing of raw NMR files (.fid)• .cnx files

Open Processor

1. Open Spectra (.fid) - Ethanol

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

2. Phase

First order phase

Zero order phase

Auto – Phase (sometimes works)

Make peaks symmetric by adjusting the sliders below (manual phasing), accept

3. Calibrate CSI (Chemical Shift/Shape Indicator)

Calibrate automatically, check DSS concentration, accept

4. Region Deletion (Water removal in versions older than 7.0)

Example: GlucoseCheck for peaks you may be deleting!!

You can click and drag the blue lines/region or type in values.No peaks inside blue area, accept!

5. Baseline CorrectionRemoves distortion in

spectrum Allows for accurate

quantification

Baseline Correction – auto linear

Baseline Correction – auto spline

• Move blue dots to centre of noise,

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

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

OPTIONAL6. Line

Line broadening: adjust Hz in

7. Shim Correction

Creates an ideal spectrum, removes lineshape distortions based

Shim correction -

8. Calibrate CSI

Calibrate CSI (Calibrate Automatically)

Congratulations, Processing completed, send to Profiler!

ChenoMX Profiler• Compound identification• .cnx file

Type compound name here (start with DSS), make sure correct library is selected

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)

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

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

Fit clusters, note concentration of metabolite/DSS

Compound Builder• Creates library files for ChenoMX• .xcpd files

Open Compound Builder

Overlay .cnx file (ChenoMX file created by processor/profiler)

Open .cnx

Edit Compound Details: name, author, magnet frequency and concentration

Ethanol

DSS

Right click peak, add new peak to new cluster

Right click, add new peak to existing cluster

NB: Blue denotes peaks currently selected

Automatic fit, you can press this multiple times.

Message that program cannot fit any longer, ok.

Adjusting peaks manually, - matching blue peaks/red line (sum line) to match the black lines (actual spectra)

Can select and group clusters (Control + G) or right click

Success! Save.

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

Conversion from .xcpd XML file

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>*

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,

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,

BAYESIL

Automated

Human

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

NMRlib Search, sort

files by user, date, name, magnet frequency,

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.

NMRlibPick peaks

Min. peak height: deletes peaks below __

Noise factor: higher # = shows less points

lower # = shows more

Peaks picked by program, DSS is

• Peaklist: delete peaks

Adding peaks (click spectra)

1

2

Peak added!

Name clusters, save

Alternative method of creating clusters: input range (ppm)

Confirm clusters

Run fitting!

Success? Not so fast! Examine fit

1. Check fit

2. Approval 3. back

Right click HMDB0008.xml, save link as

Save, SUCCESS!

Future • Finish making xmls for all these

compounds • Profile Serum (~40 cmpds) and Urine

(200+ compounds)

Thank you! (You can wake up now)

• Dr. Wishart• Igor, Trent, Rupa, Edison• Siamak, Roman• Wishartlab

Questions?

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

%207.1.pdf