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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 !!!

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NMR Spectroscopy
Nuclear Magnetic Resonance (NMR) Spectroscopy:

Any nuclues ??

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NMR Spectroscopy

Nuclei with non zero spin NMR active nuclei !!!

NMR Spectrometer
SuperconductingMagnet withProbe OperatorConsole HostComputer

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NMR Spectroscopy (for I= nuclei)

N S N
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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


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Resonance frequency,

= B0

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At 14.10 Tesla
1H

nuclei
p p p p p p p

p p p

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Resonance frequency,

= B0

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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

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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

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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

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At 14.10 Tesla

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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


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What type of energy source does the spectrometer used?

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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


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Environment 2 600.1440 MHz

Environment 3 600.1562 MHz

To excite a given nucleus

tp 90

RF
time

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

Carrier frequency (SFO1)


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Free Induction Decay (FID)

Mixture of FIDs

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At 14.10 Tesla

Fourier transformation

Fourier transformation

Mixture of FIDs

Fourier transformation

Fourier transformation

Hz

4000
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3500

3000

2500

2000

Time domain FID

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Frequency domain Spectrum

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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,
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Chemical shifts differences

Chemical shifts differences

Chemical shifts differences

1D and 2D Electrophoresis

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

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2D NMR Spectrum

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

Chemical shifts = Coordinates


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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

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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)
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H (0 - 14)

C (0 - 220)

3D NMR Spectrum (H, N, C)

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Introduction of NMR Spectroscopy and Its Applications


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

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

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NMR Samples
Single substance ???

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NMR spectrum of Single Substance


Simple Molecule

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NMR spectrum of Single Substance


Complex molecule 1

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NMR spectrum of Single Substance


Complex molecule 2

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NMR spectrum of Single Substance


Complex molecule 3

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NMR Samples

Mixture ???

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NMR spectrum of a Mixture


Plant Extract 1

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Plant Extract 2

Trends in Biotechnology

NMR spectrum of a Mixture


Human Urine

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Introduction of NMR Spectroscopy

Why NMR technique is important? What are the latest NMR applications? NMR Applications Overview
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NMR Application Overview

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NMR Application Overview

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NMR Applications Overview

Chromatographic separations and recrystallization

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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
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NMR and Molecular Information

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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

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NMR Application Overview

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NMR and Metabonomics

NATURE
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NMR and Metabonomics

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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

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Progress into the clinic- Future directions


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
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Progress into the clinic- Future directions

NATURE 491 (2012) 384392


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Progress into the clinic- Future directions

NATURE 491 (2012) 384392


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How Do You Do Metabolomics?

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How Do You Do Metabolomics?

Spinner Sample RF Coils


TXI

RT Shim Canon

RT Shim Coils Probe

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Biofluids NMR
Human Urine

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Journal of Proteome Research

Biofluids NMR

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Statistical Analysis
PLS OSC PLS

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NMR and Kidney toxicity detection

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NATURE REVIEWS | drug discovery

NMR and Metabolic Signatures of Cancer


July 2011
Progress in Nuclear Magnetic Resonance Spectroscopy
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NMR Application Overview

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NMR and Food Analysis

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Progress in Nuclear Magnetic Resonance Spectroscopy

NMR Application Overview

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NMR and Drug Discovery

NATURE REVIEWS | drug discovery


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NMR and Drug DiscoveryScreening (STD NMR)

Progress in Nuclear Magnetic Resonance Spectroscopy


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NMR Application Overview

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In cell NMR and Protein-protein Interaction

NATURE Methods
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NMR Application Overview

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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".
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NMR and Protein

The first protein structure determined by NMR.


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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

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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

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Common labeling strategy for each class size of protein

82 kDa

50 residues ~5.5 kDa 80 residues ~9 kDa 180 residues ~20 kDa

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Journal of Magnetic Resonance 173 (2005) 193207

NMR techniques and isotope labeling

15N-edited

NOESY, 15N-edited COSY Triple-resonance multidimensional NMR

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J.J. Chou and R. Sounier, Solution Nuclear Magnetic Resonance Spectroscopy, 495-516

Small protein ubiquitin (MW= 8.6 kD)

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1H

spectrum of ubiquitin

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NMR and Protein-RNA complexes

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Progress in Nuclear Magnetic Resonance Spectroscopy

NMR and Protein-RNA complexes

Progress in Nuclear Magnetic Resonance Spectroscopy


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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

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