IPPT NMR Talk Slides - Introduction of NMR Spectroscopy and Its Applications

Introduction of NMR Spectroscopy and Its Applications
Advanced Medical and Dental Institute (IPPT) Bruker Malaysia, 10-9-2013
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Introduction of NMR Spectroscopy

What is NMR represents?
Nuclear Magnetic Resonance Spectroscopy

What does NMR detects? NMR detects
Nucleus !!!
NMR Spectroscopy
Nuclear Magnetic Resonance (NMR) Spectroscopy:
Any nuclues ??
NMR Spectroscopy
Nuclei with non zero spin NMR active nuclei !!!
NMR Spectrometer
SuperconductingMagnet withProbe OperatorConsole HostComputer
NMR Spectroscopy (for I= nuclei)
N S N
N S
N S
N S
N S
1H Nuclei) NMR Spectroscopy ( Resonance frequency, =
B0
Higher energy state aligned against/ opposed to the field
External magnetic field, Bo

The proton nuclei precess at Larmor frequencies (very close to 600 MHz)
Lower energy state aligned with the field
In 14.10 T Superconducting magnet

Resonance frequency,
= B0
At 14.10 Tesla
1H
nuclei
p p p p p p p
p p p
Resonance frequency,
= B0
At 14.10 Tesla
p
600.13 MHz
p p p p p
600.13 MHz
p 600.13 MHz p
p
600.13 MHz
600.13 MHz
p
600.13 MHz
600.13 MHz
At 14.10 Tesla
600.13 MHz 600.13 MHz
600.13 MHz 600.13 MHz 600.13 MHz
600.13 MHz
600.13 MHz
1H
nuclei
Any different?
p p p p p p p p p p p p p p p p p p
p p p
1H
nuclei
Any different?
1H
nuclei
At 14.10 Tesla
NMR Spectroscopy (1H Nuclei)

External magnetic field, Bo
Higher energy state aligned against/ opposed to the field
Radio frequency pulse Radio frequency pulse
Lower energy state aligned with the field

What type of energy source does the spectrometer used?
NMR Spectroscopy
In 14.10 T Superconducting magnet (600.130 MHz)
Larmor frequency 1 Larmor frequency 2 Larmor frequency 3
Radio frequency pulse
Radio frequency pulse Radio frequency pulse
Environment 1 600.1320 MHz

To excite a given nucleus
tp 90
RF
time
A pulse excites all the magnetic nuclei in the molecule simultaneously.
Carrier frequency (SFO1)

Free Induction Decay (FID)
Mixture of FIDs
At 14.10 Tesla
Fourier transformation
Mixture of FIDs
Hz
4000
3500
3000
2500
2000
Time domain FID
Frequency domain Spectrum
Chemical shift, in isan measured along the It is the position NMR spectrum. 1D NMR Spectrum bottom of the spectrum on delta scale (in units of parts per million, ppm).
Chemical shift,
Chemical shifts differences
1D and 2D Electrophoresis
How to distinguish between the 1D and 2D NMR Spectra?
2D NMR Spectrum
(2.08,56.7) (3.58,79.9) (5.01,107.3) (4.78,107.3)
Chemical shifts = Coordinates

2D NMR Spectrum H and C
H 4.37 H 4.36
H 4.35
(4.35,60.2)
H 4.35
(4.37,54.1)
(4.36,54.4) (4.35,50.8)
ppm
4.38
4.37
4.36
4.35
4.34
3D Triple resonance experiment on 15N,13C labeled of proteins

F2 (ppm)
C H N(c)
N (0 - 40)
N(b)
F1 (ppm)
N(a)
F3 (ppm)
H (0 - 14)
C (0 - 220)
3D NMR Spectrum (H, N, C)
Introduction of NMR Spectroscopy and Its Applications

Advanced Medical and Dental Institute (IPPT) Bruker Malaysia, 10-9-2013
NMR Samples Any kind of sample? Pure substance or mixture ?
NMR Samples
Single substance ???
NMR spectrum of Single Substance

Simple Molecule

Complex molecule 1

Complex molecule 2

Complex molecule 3
NMR Samples
Mixture ???
NMR spectrum of a Mixture

Plant Extract 1
Plant Extract 2
Trends in Biotechnology
NMR spectrum of a Mixture

Human Urine
Introduction of NMR Spectroscopy
Why NMR technique is important? What are the latest NMR applications? NMR Applications Overview
NMR Application Overview
NMR Applications Overview
Chromatographic separations and recrystallization
Conventional Spectroscopic Methods

IR spectroscopy High resolution MS NMR spectroscopy
OH HO OH O
2 3 1 4 19 9 10 5 11 12 13 21
O
20 8 7 14
OH OH
15 16
H
6
O H
H
18 Innovation with Integrity
NMR and Molecular Information
OH HO OH O
2 3 1 4 19 9 10 5 11 12 13 21
O
20 8 7 14
OH OH
15 16
H
6
O H
H
18
NMR and Metabonomics
NATURE
NMR and Metabonomics
Progress into the clinic- Future directions
metabonomics is used to probe the real-world nature of biochemical functionality and is sensitive to both gene and environmental influences; it is, therefore, likely to be more practical than gene-based measurements of responses to therapy. we affirm that metabonomics has the power to influence clinical decision-making in the hospital environment for both medical and surgical treatments. NATURE 491 (2012) 384392

NMR-based metabonomics of biofluids offers great promise for the minimally-invasive screening of cancer related perturbations, at several stages of disease management, ranging from early detection and differentiation from benign conditions to identification of metastatic disease and surveillance of recurrence. metabolic profiling of biofluids through NMR spectroscopy will, in time, provide a unique and important tool in the clinical management of cancer, and have major impacts in areas such as screening, diagnosis and monitoring of disease progression. metabonomics studies have great potential in the emerging area of personalized cancer therapy, namely through the identification of metabolic markers of treatment efficacy or early toxicity, thus assisting in the development of novel, targeted therapeutics Progress in Nuclear Magnetic Resonance Spectroscopy 62 (2012) 5174
NATURE 491 (2012) 384392

NATURE 491 (2012) 384392

How Do You Do Metabolomics?
How Do You Do Metabolomics?
Spinner Sample RF Coils

TXI
RT Shim Canon
RT Shim Coils Probe
Biofluids NMR
Human Urine
Journal of Proteome Research
Biofluids NMR
Statistical Analysis
PLS OSC PLS
NMR and Kidney toxicity detection
NATURE REVIEWS | drug discovery
NMR and Metabolic Signatures of Cancer

July 2011
Progress in Nuclear Magnetic Resonance Spectroscopy
NMR and Food Analysis
NMR and Drug Discovery
NATURE REVIEWS | drug discovery

NMR and Drug DiscoveryScreening (STD NMR)

In cell NMR and Protein-protein Interaction
NATURE Methods
Nobel Prize in Chemistry 2002
In 2002, The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry to Prof Kurt Wthrich "for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution".
NMR and Protein
The first protein structure determined by NMR.

J. Mol. Biol.
Protein structure determination categorized by experimental method Protein Structure Deposited in Protein Data Bank as of July 2013
626 10.8 % 9955 60 188
X-ray NMR
88.3 % 81638
Electron Microscopy Solid state NMR Other
The growth in the number of NMR-deduced protein structure

Protein Structure Deposited at Protein Data Bank Based on Solution NMR as of July 2013
4000 3500 3000 2500 2000 1500 1000 500 0
Before 2000 2000-2005 2005-2010 2010-Today
Common labeling strategy for each class size of protein
82 kDa
50 residues ~5.5 kDa 80 residues ~9 kDa 180 residues ~20 kDa
Journal of Magnetic Resonance 173 (2005) 193207
NMR techniques and isotope labeling
15N-edited
NOESY, 15N-edited COSY Triple-resonance multidimensional NMR
J.J. Chou and R. Sounier, Solution Nuclear Magnetic Resonance Spectroscopy, 495-516
Small protein ubiquitin (MW= 8.6 kD)
1H
spectrum of ubiquitin
NMR and Protein-RNA complexes
NMR and Protein-RNA complexes

Sensitivity of NMR
Compared with other analytical techniques, NMR is significantly insensitive in terms of the minimum sample amounts for an analysis. The limit of detection (LOD) of NMR Laser-induced fluorescence FT IR and Raman spectroscopy Mass spectrometry
109 - 1011 mol 1013 mol, 1012 - 1015 mol 1019 mol
www.bruker-biospin.com

IPPT NMR Talk Slides - Introduction of NMR Spectroscopy and Its Applications

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Introduction of NMR Spectroscopy and Its Applications

Advanced Medical and Dental Institute (IPPT) Bruker Malaysia, 10-9-2013

Innovation with Integrity

Introduction of NMR Spectroscopy

Nuclear Magnetic Resonance Spectroscopy

Innovation with Integrity

Innovation with Integrity

Nuclei with non zero spin NMR active nuclei !!!

Innovation with Integrity

NMR Spectroscopy (for I= nuclei)

1H Nuclei) NMR Spectroscopy ( Resonance frequency, =

External magnetic field, Bo

Lower energy state aligned with the field

In 14.10 T Superconducting magnet

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Innovation with Integrity

Innovation with Integrity

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600.13 MHz 600.13 MHz

600.13 MHz 600.13 MHz 600.13 MHz

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NMR Spectroscopy (1H Nuclei)

Radio frequency pulse Radio frequency pulse

Lower energy state aligned with the field

What type of energy source does the spectrometer used?

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Larmor frequency 1 Larmor frequency 2 Larmor frequency 3

Radio frequency pulse

Radio frequency pulse Radio frequency pulse

Environment 1 600.1320 MHz

Environment 2 600.1440 MHz

Environment 3 600.1562 MHz

To excite a given nucleus

A pulse excites all the magnetic nuclei in the molecule simultaneously.

Carrier frequency (SFO1)

Free Induction Decay (FID)

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Time domain FID

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Frequency domain Spectrum

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Chemical shifts differences

Chemical shifts differences

Chemical shifts differences

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How to distinguish between the 1D and 2D NMR Spectra?

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(2.08,56.7) (3.58,79.9) (5.01,107.3) (4.78,107.3)

Chemical shifts = Coordinates

2D NMR Spectrum H and C

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3D Triple resonance experiment on 15N,13C labeled of proteins

3D NMR Spectrum (H, N, C)

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Introduction of NMR Spectroscopy and Its Applications

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NMR Samples Any kind of sample? Pure substance or mixture ?

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NMR spectrum of Single Substance

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