NMR Introduction To Biological Sample

Biol. Rev. (2010), pp. 000000.
doi: 10.1111/j.1469-185X.2010.00157.x
An introduction to biological nuclear magnetic

resonance spectroscopy
John H. F. Bothwell1,2,3 and Julian L. Griffin4
1 Medical
Biology Centre, Queens University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK
Marine Biological Association of the UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK
3 UMR 7139, Station Biologique, Place Georges Teissier, 29682, Roscoff Cedex, France
4 The Hopkins Building, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK
2
(Received 26 April 2009; revised 30 July 2010; accepted 10 August 2010)
ABSTRACT
Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology.
This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in
acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather
than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to
address a number of biological problems. Using detailed examples, we discuss the use of 1 H NMR spectroscopy in
mixture analysis and metabolomics, the use of 13 C NMR spectroscopy in tracking isotopomers and determining the
flux through metabolic pathways (fluxomics) and the use of 31 P NMR spectroscopy in monitoring ATP generation
and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe
the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how
it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms.
We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent
advancessuch as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisationare
opening new avenues for todays biological NMR spectroscopists.
Key words: 13 C, 1 H, hyperpolarization, magic angle spinning, magnetization transfer, metabolomics, multidimensional
NMR, NMR spectroscopy, 31 P, pulse sequences.
CONTENTS
I.
II.
III.
IV.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Running an NMR experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(1) Measuring intensity and optimizing the signal-to-noise ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(a) Increasing B0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(b) Signal averaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(c) Increasing the population difference in a sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(d) Temperature reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(e) Volume reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(2) Measuring energychemical shift, or . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(a) Shimming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(b) Sample spinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(c) Solution-state studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(d) Magic angle spinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6
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8
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* Address for correspondence: (Tel: +44 (0)28 90972269; Fax: +44 (0)28 90975877; E-mail: j.bothwell@qub.ac.uk or jhbot@mba.ac.uk)
Biological Reviews (2010) 000000 2010 The Authors. Biological Reviews 2010 Cambridge Philosophical Society
John H. F. Bothwell and Julian L. Griffin
V.
VI.
VII.
VIII.
(3) Measuring relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(4) Measuring phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis, or what can the frequency domain tell us? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(1) How much is there? Metabolic foot- and finger-printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(2) What is it? Metabolic profiling and protein NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(a) Chemical shift and spin-spin coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(b) Multidimensional NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(c) Hyphenated NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(3) Where is it? Metabolite environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(4) What is it doing? Metabolite kinetics/behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. INTRODUCTION
Nuclear magnetic resonance, or NMR, spectroscopy uses
radiofrequency waves to reveal information about magnetic
nuclei. Since the word spectroscopy describes any technique
in which electromagnetic (EM) radiation is used to probe
atoms (Figure 1), NMR spectroscopy is only one of a range
of spectroscopic methods in everyday biological use.
However, for reasons which we will consider herein, NMR
differs from other forms of spectroscopy in three important
ways. First, NMR looks at how the nuclei of a specific,
user-selected, chemical element are distributed amongst the
molecules of a sample, giving NMR a broader range of
targets than most spectroscopic techniques. Second, NMR
signals are sensitive to the local surroundings of the nuclei
under observation, providing a tool that can probe the
chemical and physical environment of an atom and which
can reveal more information about a given sample than
most other spectroscopic techniques. Third, NMR is more
highly penetratingbut, usefully, less damagingthan
other forms of spectroscopy. Why is NMR spectroscopy
so powerful?
To answer this question, imagine standing at the Earths
North Magnetic Polecurrently an ice floe in the Canadian
Arcticwith all of the Worlds compasses pointing towards
us. Their needles can, with a little effort, be forced to point
in another direction, but are in their most stable states when
aligned with the Earths magnetic field and will relax back
to pointing north as soon as they are released. A number
of common biological elements have nuclei which behave
much like these compasses; they will align themselves in a
magnetic field, may be forced to point in another direction,
and will relax back to point north once released, usually
within a few hundred milliseconds.
There are two things which we need to add to this
magnetic compass analogy to understand enough NMR
for most biological applications. First, a magnetic compass
can be deflected to point in any direction, but quantum
mechanical laws restrict magnetic nuclei to pointing in a
much more limited set of directions. These directions are
actually nuclear energy levels called spin states where spin
refers to the fact that the magnetism of a given nucleus may be
10
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11
12
12
13
14
14
14
15
16
16
considered to arise from the spinning motion of its electrical

charge. For instance, when the 1 H and the 13 C nuclei are in
a magnetic field, they each have only two allowed spin states,
which are called + 1/2 (spin up) and 1/2 (spin down). 1 H and
13 C are therefore known as spin 1/ nuclei. It is important
2
to realize that, in any population of 1 H and 13 C nuclei, not
all of the nuclei will be in the same spin state; some will be
in the higher energy, excited, + 1/2 state, the rest will be in
the lower energy, ground, 1/2 state, with their proportions
depending on the energy difference between the two spin
statesthe higher the energy difference, the more nuclei will
be in the 1/2 state. Other nuclei, known as quadrupolar
nuclei snd including the biologically informative 2 H, 18 O
and 23 Na, have spins >1/2 and will also have detectable
NMR signals. It is, however, important to remember that,
although most elements have at least one magnetic isotope,
not all nuclei are magnetic and only those with non-zero soin
will give a detectable NMR signal.
The second point which we need to add to our
compass analogy is that individual magnetic nuclei may
be moved, or flipped, from one spin state to another
by radiofrequency (RF) waves whose exact frequencies are
diagnostic for the chemical element involved. This RF-waveinduced flipping of magnetic nuclei was first observed over
70 years ago by Isidor Rabis group at Columbia University
(Rabi, 1937; Rabi et al., 1938) and named nuclear induction.
However, because some of the early theories to describe this
nuclear flipping employed the idea of nuclei resonating at the
frequencies at which they absorbed RF waves, Rabis nuclear
induction quickly became known as nuclear magnetic
resonance, or NMR (Gorter & Broer, 1942). We will refer
to NMR spectroscopy throughout this review, although it
should be noted that, in a clinical context, it is common
practice to drop the n-for-nuclear to avoid troubling patients
unnecessarily with unfounded fears of nuclear radioactivity.
Under clinical conditions, therefore, NMR spectroscopy is
usually shortened to magnetic resonance spectroscopy, or
simply MRS.
Following Rabis initial physics-based discoveries, NMRs
biological applications began to be realized in 1954,
when a group at Stanford used 1 H NMR spectroscopy
to show that DNA strands have large hydration shells
Biological NMR spectroscopy
3
audience which is interested in the biological applications
and potential of NMR spectroscopy, but which lacks the
mathematical background to tackle traditional theoretical
approaches to the subject. We will start by looking at the
routines involved in running a typical NMR experiment, we
will then explain how these routines reflect the physical basis
of NMR and we will end by demonstrating what information
these routines can give us.
This review has three main sectionsthe first two
(Sections III and IV) are a brief introduction to the principles
behind running an NMR experiment, the third (Section V)
is a more detailed look at the information which can
be obtained from NMR spectroscopy. For those who
wish to read more, we recommend the following basic
(Claridge, 1999; Derome, 1987; Hore, 1995) and advanced
(Hore, Jones & Wimperis, 2000; Keeler, 2005; Levitt, 2001)
textbooks, all of which are excellent.
Fig. 1. Some physical properties of the elctromagnetic

spectrum. (A) Electromagnetic (EM) radiation is packaged into
photons. Each photon has an associated frequency, , and
wavelength, . Matter can only absorb EM radiation at discrete
wavelengths, because the energy, E, of any beam of EM
radiation is related to its wavelength, , by a constant, h, in
the formula E = h/. (B) Different regions of the EM spectrum
are used for different types of spectroscopy. UV, ultraviolet;
Vis visible; IR, infrared; RF, radiofrequency; NMR, nuclear
magnetic resonance.
(Jacobson, Anderson & Arnold, 1954). Only three years

later, biomolecular NMR had advanced to the stage where
an entire intact protein, ribonuclease, was being examined
(Saunders, Wishnia & Kirkwood, 1957) and, today, NMR
spectroscopy has become one of the most powerful and
flexible analytical techniques available to biologists, being
one of the few methods which give analytical information
from deep within living tissue. The solid theoretical
foundations of the effect have been exploited in innovative
techniques such as multi-dimensional spectroscopy (Kumar,
Welti & Ernst, 1975), Magic Angle spinning of solids (Li,
2006; Lowe, 1959) and quantum computing (Cory, Fahmy
& Havel, 1997), while high-throughput NMR is increasingly
driving post-genomic functional studies (Bundy et al., 2007).
This makes it a regrettable, and preventable, shame
that many biologists still view NMR as a prohibitively
mathematical technique. Not only is NMR grounded
in a phenomenon which most biologists use without
demur in other contextsspectroscopy using EM radiation
(Fig. 1B)but many of the applications of NMR can be
understood in non-mathematical terms. Indeed, modern
NMR spectrometers allow their users to acquire and
analyze complex spectra without needing any deep physical
understanding of the concepts by which the data are
generated.
For these reasons, and because of the ever-increasing
workload which modern -omics and structural biology
place on high-throughput analytical techniques, we have
written this review for the large professional and student
II. RUNNING AN NMR EXPERIMENT

NMR experiments are, in practical terms, fairly straightforward. For simplicitys sake, we will break them down into
three partssample preparation, data acquisition and data
analysiseach of which will be covered in separate sections
of this review.
During sample preparation (Section III), the tissue of
interest is prepared, most commonly as a solution in a
narrow-walled glass tube, and put into a high-field electromagnet whose large coil generates a strong B0 pronounced
B noughtfield (Section IV). Once inside the B0 magnet,
the sample sits in a second, smaller, electromagnetic coil
which is found at one end of a poster-tube-sized cylindrical
probe. This second coil is, understandably, known as the
probe coil (Section IV).
During data acquisition (Section IV), the samplestill
in the magnets B0 fieldis irradiated with RF waves
generated by the probe coil. RF waves have electrical and
magnetic components, so these irradiating RF waves are
often known by their magnetic name, which is the B1 field.
These RF wavessometimes grouped together in a cluster
called a pulse sequence (Sections IV.1.c and V.2.b)flip
nuclei in the sample from one spin state to another. The
more power applied in the RF wave, the more nuclei
are flipped, a process known as saturation (Section IV.1.c).
The irradiating RF waves/B1 field are then turned off
and the nuclei allowed to relax (Section IV.3) back to
their equilibrium population, which they do by emitting
RF waves whose parameters provide information about
the sample. These emitted RF waves are collected as
Free Induction Decays, or FIDs (Section IV.3) in the
Time domain (Section IV.3). The FIDs are mathematically
simplified and transferred into the Frequency domain, using
a technique called Fourier Transformation (Section IV.4),
where they are usually presented in the form of a onedimensional (1D) or two-dimensional (2D) spectrumor
transientcontaining a number of resonances (Section IV
4
A
Fig. 2. Sample one-dimensional (1D) and two-dimensional

(2D) nuclear magnetic resonance (NMR) spectra of yeast
metabolite extracts. (A) A 1D 1 H NMR spectrum of yeast
metabolites, showing intensity on the y axis and resonant
frequency (also known as or chemical shift, see Section IV.2a)
on the x axis. (B) A 2D spectrum of the same sample as in A, with
both axes now representing a different resonant frequency 13C
for the y axis and 1 H for the x axisand the intensity of the
peak being reflected by the closeness of packing in the contour
plot. It should be noted that the y and x axes are often called
by their spectroscopic names of F1 and F2, respectively. This
spectrum comes from a heteronuclear single quantum coherence
(HSQC) experiment, which is used to follow 1 H nuclei which
are attached to 13 C nuclei. We thank Reza Salek, Duncan
MacInnis and Juan Castrillo at the University of Cambridge for
allowing us to present these spectra.
and Fig. 2). The emitted RF radiation has four parameters

(Fig. 3): (1) intensity (Sections IV.1 and V.1), (2) frequency,
which is more commonly expressed as chemical shift
(Section IV.2.a), (3) half-life (Sections IV.3 and V.3) and
(4) phase (Sections IV.4 and V.2b) and in Section V we will
look in turn at how an NMR experiment extracts information
from each of these parameters.
III. SAMPLE PREPARATION

One of the most striking things about NMR is its ability
to acquire information non-invasively and non-destructively
from an exceptionally broad range of samples, whether in vivo
from within living organisms, such as the human body (Hoult
et al., 1974), ex vivo from isolated organ preparations (Bittl
& Ingwall, 1985; Brindle & Radda, 1985) and intact tissue
samples, such as tumours (Griffin et al., 2003a), or in vitro from
homogenous tissue extracts in solid, liquid or gaseous form.
NMR spectroscopy displays this versatility because it involves
nuclear absorption and emission of RF radiation. Since the
Fig. 3. Building free induction decays (FIDs) and onedimensional (1D) spectra from the four common nuclear
magnetic resonance (NMR) parameters. (A) Nuclei (filled circles)
which have been excited to a high energy spin state will relax
back over time (x axis) to a lower energy, ground, state through
the emission of photons of electromagnetic (EM) radiation
(wavy lines). (B) When these EM photons are summed over
a population of relaxing atoms, the decay in the intensity
of emitted EM radiation over time will produce a FID.
FIDs may be described using four parameters: (1) intensity,
(2) frequency, (3) half-life, or t 1/2, and (4) phase. NMR is one of
the few spectroscopic techniques which allows information to be
extracted from all four of these parameters. RF radiofrequency.
(C) Using a technique called Fourier transformation, the FID,
or plot of intensity versus time in the time domain, is converted
into a plot of intensity versus frequency in the frequency
domain.
nucleus makes up only a very small part of any atom, RF

radiation directed into tissue will have only a small chance of
hitting a nucleus and so RF waves will tend to penetrate tens
of centimetres into tissue before they are absorbed, allowing
both imaging and spectroscopy inside the human body for
example. Furthermore, because the flipping of a magnetic
nucleus from one spin state to another involves only a very
small amount of energy, any tissue irradiated by RF radiation
will suffer very little damage from subsequent heating as
nuclei relax back into their original ground state. A technique
such as ultraviolet (UV) spectroscopy, by contrast, involves
the absorption and emission of radiation by the electron
clouds which make up the bulk of an atoms volume, so UV

radiation is absorbed rapidly and cannot penetrate as deeply
into tissue as RF radiation. Similarly, were microwaves used
then they would be absorbed and released by the covalent
bonds between atoms, leading to increased molecular motion
and significantand damagingsample heating.
Nonetheless, despite this enormous potential for in vivo
work, the bulk of NMR spectroscopy is carried out on
samples in solution, for reasons which we will consider in
Section IV.2c. Some ex vivo samplessuch as blood, urine
or cerebrospinal fluidare already in solution and may
be observed almost directly, although care must be taken to
ensure that these samples are not compromised in other ways.
The haemoglobin in blood, for instance, is paramagneticit
becomes magnetic in a magnetic fieldand so can distort
the applied B0 field enough to broaden resonances in the
NMR spectrum to an extent where they overlap and much
meaningful information is lost, for reasons discussed in
Section IV.2. Blood, therefore, is usually analysed as serum or
spun down to remove the haemoglobin-containing red blood
cells and leave plasma. When this is done, the osmolarity of
the serum or plasma must be maintained with salts during
any dilution to preserve structures such as lipoproteins. Other
fluids present their own idiosyncrasies; NMR spectroscopy of
urine can be impaired by the presence of proteins and metal
ions such as Mg2+ and Ca2+ which bind various metabolites
and broaden their resonances. To overcome this, one can
use chelating agents such as ethylenediaminetetraacetic acid
(EDTA), which bind these cations (Nicholson, Buckingham
& Sadler, 1983). In addition, the pH of urine samples
must be fixed by balancing the ratio of sodium phosphate
(NaH2 PO4 ) to orthophosphate (Na2 HPO4 ) to prevent the
chemical shift of certain resonances varying from sample to
sample (Beckwith-Hall et al., 1998).
For in vitro tissue extracts, of course, solubilization is much
less of a problem and sample preparation is able to focus
on the efficient extraction of any molecules present. This
is made easier because, in contrast to methods such as
gas chromatography mass spectrometry (GC-MS), NMR
samples do not need chemical derivatization to make them
detectable. A range of extraction methods are available
and have previously been summarized (Le Belle et al.,
2002), with a typical extraction protocol reading something
like this:
Frozen [rat brain] slices were transferred to a mortar
kept on dry ice. The slices were ground to a powder, and
suspended in 5 mL of ice-cold 6% perchloric acid. This
suspension was spun at 1500 g for 5 min and the supernatant removed [. . .], neutralized with 1 M KOH and
freeze-dried. The freeze-dried residue was resuspended
in 650 l of deuterated water (D2 O) containing 2 mM
3-trimethylsilyl-deuterosodium propionate (d 4 -TSP) as
a reference standard. (Bothwell et al., 2001, p1634).
Perchloric and trichloric acids are a popular choice for
aqueous extracts, especiallyas in this examplefor brain
tissue in which metabolism must be quenched rapidly. However, acid extraction has the unfortunate disadvantage of
oxidizing many metabolites and proteins, so alternative
extraction protocols are also used (Pears, 2007). The most
common of these is chloroform/methanol extraction, with
other widely used procedures including acetonitrile/water
extraction and methanol/ethanol/water extraction. These
alternatives have the advantage of partitioning metabolites
among several distinct fractionsusually a polar, aqueous
metabolite fraction, a non-polar, lipophilic metabolite fraction and an intact and unoxidized protein pelletallowing
more complete extraction of metabolites and better characterization of the sample (Fig. 4).
As in the example protocol, above, extracted fractions
are usually diluted intoor freeze-dried and reconstituted
Fig. 4. Different nuclear magnetic resonance spectra from

different fractions from a biological tissue. (A) A chloroform/methanol extraction produces three fractions. (B) The
lower chemical shift region of the 1 H NMR spectrum from the
aqueous, methanol fraction of extracts from the Hwacheong
rice cultivar (Oryza sativa L.). This region of the spectrum
contains aliphatic polar metabolites, including (1) Trigonelline,
(2) overlapping sugar resonances, mostly from sucrose, fructose
and glucose, (3) glycerophosphocholine, (4) O-acetyl carnosine, (5) asparagine, (6) methionine, (7) glutamine, (8) glutamate,
(9) acetate, (10) lysine, (11) alanine, (12) lactate and (13) overlapping resonances from leucine, isoleucine and valine. (C) Any
protein in the sample is less dense than chloroform and forms
a layer between the chloroform and aqueous phases. (D) The
lower chemical shift region of the 1 H NMR spectrum from the
corresponding organic, chloroform fraction extracted from the
same sample as B. This fraction contains non-polar metabolites,
including resonances from unsaturated fats (15, 16, 19), saturated fats (17, 18), common to all fatty acids (20) and unidentified
metabolites too close to the baseline (14). We thank Oliver Jones
at the University of Cambridge for allowing us to present these
spectra.
6
intosolvents in which protons (1 H) have been replaced
by deuterons (2 H). Deuterons produce NMR signals at a
different frequency to those of protons, so these deuterated
solvents serve two purposes. First, they lower the solvents
contribution to the observed 1 H spectrum by reducing
the amount of protonated solvent, which is a far from
trivial effect when we consider that the concentration of
water-as-solvent is usually around 50 mol l1 , as opposed to
millimolar metabolite concentrations. Solvent effects can also
be removed by simple solvent suppression pulse sequences,
as explained in Section IV.1b.
Second, most modern NMR spectrometers are operated
in a deuterium locked mode where the known frequency of
the deuterium signal is used to calibrate the other frequencies
observed during the NMR experiment. Deuterium locking
is usually supplemented by adding a chemical shift reference
such as, for 1 H NMR spectra, 3-trimethylsilyl-deuterosodium
propionate (TSP) or Tetramethylsilane (TMS) to define more
accurately chemical shifts (Harris et al., 2002). It should
be noted that deuterium locking is less common in older
NMR studies, in which external reference standards are
used exclusively; this is because older machines were unable
to detect the deuterium signal at the same time as the
observed nucleus or, for protein structure determination, the
observed nuclei.
In our example, above, tissue was reconstituted into
deuterated water (D2 O), but many other solvents are also
available: deuterated chloroform (CDCl3 ) is commonly used
for lipid metabolites, deuterated di-methyl sulfoxide (DMSO)
has been used to solubilise components in the humin fraction
of soil (Simpson et al., 2007) and a range of deuterated solvents
are used during combined liquid chromatography and NMR
spectroscopy experiments (Section V.2c), according to the
chromatographic separations that are being used (Dunn,
Bailey & Johnson, 2005).
We have emphasized sample preparation for one very
good reason: so long as an NMR spectrometer is configured
and operated correctly, multiple runs on the same sample
show an exceptionally high degree of reproducibility,
making high-resolution NMR spectroscopy a more robust
analytical approach than those based on chromatographic
and/or mass spectrometry based methods, such as high
performance liquid chromatography (HPLC) or GC-MS.
In practical terms, this means that, so long as a standard
operating protocol is followed, researchers can be confident
that variation between samples reflects biological, and not
instrumental, variation. This is particularly important if
spectra are to be analysed by automated pattern recognition
tools. Indeed, NMR is such a powerful global analytical
approach that it can often detect unexpected biological
variation in a dataset and variations in diet, age, growth
conditions, hormonal status and strain background have
all unexpectedly muddied the interpretation of studies in
plants, animals and micro-organisms (Bollard et al., 2005;
Griffin & Nicholls, 2006; Gulston et al., 2008). The more
standardized the sample preparation can be made, therefore,
the better.

IV. DATA ACQUISITION
After preparation, samples are put into the most distinctive
feature of an NMR experimentthe barrel-shaped
aluminium magnetand data acquisition begins. The
following example protocols give an idea of the various
magnet and spectrometer settings which must now be
considered:
All spectra were obtained at 30 C on a [. . .] spectrometer operating at a proton frequency of 400.15 MHz.
Fully relaxed spectra were acquired with 90 pulses
applied every 13s for 512 transients. The decoupler was
gated on the water frequency, in the delay between pulses,
to suppress the residual water peak. (Bothwell et al.,
2001, p1634).
1H spectra were acquired at 400 MHz into 40,000
data points using a 90 pulse, an acquisition time
of 4 s, and a sweep width of 5 kHz. The overall pulse
repetition time was 5 s. The samples were spun at 16 Hz
and maintained at 30 C during data acquisition. The
spectra were the sum of 128 transients. (Raamsdonk
et al., 2001, p 50).
As we stated at the end of Section II, the purpose of these
protocols is to measure four parameters (intensity, frequency,
half-life and phase; Fig. 3) of the EM radiation which is
released when a population of nuclei relax from one spin
state to another. We will look in turn at how an NMR
experiment measures each of these parameters.
(1) Measuring intensity and optimizing the
signal-to-noise ratio
Any spectroscopic technique benefits from a high signal-tonoise ratio and an important limitation with NMR, relative
to other spectroscopies, is its lack of sensitivity. The inherent
ability of any nucleus to absorb EM radiation is given by its
gyromagnetic ratio: nuclei with higher gyromagnetic ratios
will absorb EM radiation more readily than nuclei with lower
gyromagnetic ratios.
Theorists in the late 1920s and early 1930s realized that
the energy difference between nuclear spin states would
depend on the strength of the magnetic field in which the
nuclei were sitting. Unfortunately, in the Earths magnetic
field, these energy differences are vanishingly small, so that,
even for nuclei with higher gyromagnetic ratios, the small
energy differences between nuclear spin transitions are of the
order of thermal energy at room temperature. This means
that ambient heat suffices to move many nuclei into higher
spin states, making the population difference between high
and low energy spin states very smalltypically less than a
few parts per million. This, in turn, reduces the number of
low energy nuclei which may be excited in a sample and thus
reduces the maximum achievable signal-to-noise.
To overcome this low-sensitivity problem, spectroscopists
use five common methods to increase the signal-to-noise
ratio for a given mass of sample: (a) they increase B0 ,

(b) they average together many signals, (c) they increase the
saturation of the sample, (d) they reduce the temperature
of the sample and (e) they reduce the volume in which the
NMR signal is observed.
(a) Increasing B0
Since theory predicted that energy differences between
nuclear spin states varied with the external magnetic field
strength, and since very low energy EM radiation was
almost impossible to detect using the electronics available
in the 1930s, Isidor Rabis pioneering group at Columbia
placed nuclei in an extremely strong homogenous B0 field.
This increased the energy differences between spin states
so that the nuclei could absorb EM radiationapplied
as a B1 fieldin the more easily detectable RF range.
These early experiments used molecular beams of nuclei,
because these early workers were interested in nuclear
physics, not in probing biological samples. However, Edward
Purcell (Purcell, Torrey & Pound, 1946) and Felix Bloch
(Bloch, Hansen & Packard, 1946), working independently at
Stanford and MIT, soon realized that NMR signals could
also be obtained from more everyday preparations. Paraffin
wax and tubs of water were early favourites; we have already
explained (Section III) that the strong 1 H signal generated
by water remains one of the most common complications in
biological NMR spectroscopy and is the reason why samples
are usually reconstituted in deuterated solvents.
Magnetic field strength is usually measured in Tesla, given
the symbol T. Todays B0 fields are generated by powerful
electromagnets which generate fields between 2 and 21 T,
several hundred times as strong as the Earths own 3060 mT
(Le Mouel, Kossobokov & Courtillot, 2005) field. The actual
electromagnetic coil takes up only a small amount of the
distinctive aluminium barrels, but in order to generate such
high B0 fields, superconducting material must be used and
this, in turn, must be cooled to liquid helium temperatures
of around 4 K (269 C). The bulk of the barrel consists,
therefore, of two cooling jacketsa liquid helium one to
cool the electromagnet and a liquid nitrogen one to cool
the liquid helium. Furthermore, in modern magnets the
superconducting B0 coil is surrounded by another coil which
prevents, at least in part, the B0 field from extending outside
the cylinder, so that spectroscopists can hug a 9.4 T magnet
and still be certain that their credit cards will survive.
The first way of improving signal-to-noise would simply
be to increase the strength of the B0 field, to a current
commercial maximum of 21 T, or 950 MHz, thereby
increasing the population difference between low and high
spin states. Unfortunately, this is usually a prohibitively
expensive way to increase signal-to-noise ratios, as magnet
costs increase rapidly with field strength.
It should be noted that there is an alternative measure
of magnet strength commonly used by spectroscopists.
We mentioned in Section I that nuclei which absorb RF
radiation of a particular frequency are said to resonate
at that frequency, hence the R-for-Resonance in NMR
and hence why absorption peaks are commonly known as

resonances. As we have explained, the EM frequency at
which nuclei absorb, or resonate, depends upon the strength
of the B0 field; in a 9.4 T magnet, for example, 1 H nuclei
resonate at 400 MHz, in the same range as wireless local area
network (LAN) and mobile phones, and 13 C nuclei resonate
at 100.2 MHz. Magnets are, therefore, often described by
the frequency at which 1 H nuclei resonate, so that the same
magnet may equally correctly be referred to as a 9.4 T
magnet or a 400 MHz magnet, according to taste.
(b) Signal averaging
Second, and very commonly, signal-to-noise ratio can be
improved by averaging together individual spectra, or
transients. However, while signal increases as a function
of
the number of transients, n, noise increases as a function
of n, which
means that the signal-to-noise ratio increases as
n/ n, or n. A two-fold increase in signal-to-noise therefore
comes at a four-fold cost in time, which can soon become
prohibitive.
(c) Increasing the population difference in a sample
In order to maximize signal-to-noise in, for example, NMR
spectroscopy with spin 1/2 nuclei such as 1 H and 13 C, the
excess nuclei in the ground, 1/2, state should be flipped into
the + 1/2 state to give equal populations of nuclei in each spin
state. This process of population equalization is called full
saturation of the sample and is achieved by applying a 90
pulse from the B1 coil; 90 reflects the fact that when we
redistribute magnetic nuclei in this way, we also move the
net magnetization of the sample from the z axis to the xy
plane, i.e. through 90 .
After saturation, we need to collect all of the emitted
RF signal by waiting for the flipped + 1/2 nuclei to relax
fully back to the ground 1/2 state, restoring the original
population difference between spin states. Unfortunately, it
takes a long time to collect spectra using both full saturation,
using a 90 pulse, and full relaxation, using a betweenpulse delaythe relaxation delaylong enough to allow
full restoration of the original populations. This is is a
particular problem if many transients are being averaged
together. For these reasons, spectroscopists usually aim to
strike an optimal balance between minimizing run time and
maximizing signal-to-noise, and they do this by applying
less-than-90 RF pulses (often around 60 ) and allowing
only partial relaxation. Most NMR spectra are, therefore,
acquired under partial saturation and partial relaxation. For
most purposes, partial saturation is beneficial, although it can
complicate the quantification of some compounds, especially
those with long relaxation times, such as the carbonyl 13 C
nuclei (R2 -13 C=O) found in carboxylic acids.
As an aside, saturation is also used to increase signal-tonoise ratio by reducing noise; specifically, noise from solvent
protons (Section III). Because the water resonance is so large,
it can swamp other resonances and so is removed from the
spectrum by irradiating the water resonance continually, so
8
that the population difference between high and low energy
states is zero. This means during any subsequent irradiation
by EM radiowaves there are no protons in water molecules
to contribute a net magnetisation for the NMR effect. The
water resonance has effectively been decoupled from the
sample resonances, so this process is known as decoupling.
Finally, we can see that decoupling is the simplest example
of a pulse sequence, since it involves using one RF pulse
to suppress water and then a second RF pulse to excite the
sample nuclei-of-interest. We will meet more complex pulse
sequences later (Section V.2b).

Even without the improvements associated with
cryoprobes and microprobes, typical sample acquisition
times for a 1 H NMR experiment where the sample is
not limited are between 6 and 15 min. Furthermore, the
approach is cheap on a per sample basis and thus large
datasets can be rapidly acquired, especially if automation is
used, with more than 150 samples in 24 hours being easily
achievable. Hence, the tool has been widely used in the drug
safety assessment area of toxicology during drug screening
(Beckwith-Hall et al., 1998).
(2) Measuring energychemical shift, or
(d) Temperature reduction

The signal-to-noise ratio can be increased by temperature
reduction in two different ways. Either the temperature of
the sample may be reduced by coolingalthough this is
limited by the temperature at which the solvent freezesor
the temperature of the equipment may be reduced using
cryogenic probes (Styles et al., 1984). Sample cooling reduces
thermal motion so that more nuclei will sit in lower energy
spin states. This increases the population difference between
ground and excited spin states and thus increases signal-tonoise ratio by increasing the maximum signal. Probe cooling,
on the other hand, reduces the contribution of electronic
and thermal noise and provides a theoretical up-to-four-fold
increase in signal-to-noise ratio. The increased sensitivity of
cryogenic probes has the added benefit of allowing the use
of nuclei with lower sensitivity (i.e. lower gyromagnetic ratio)
than 1 H, which would normally be prohibited due to the
time required to acquire a sufficient signal (Keun et al., 2002).
(e) Volume reduction
Finally, the more homogenous the B0 field, the better the
signal resolution. Since a homogenous field is easier to
generate over a small volume than a large, most magnets
have narrow bores of only a few centimetres, although
those used for in vivo work must, of course, have bores large
enough for living organisms to fit inside. These larger bores
range from tens of centimetres for small animals to the wide
bore medical magnets used for patient studies. Provided
the individual is stationary, the effect of magnetic fields on
patients is minimal, although recently the EU has tried to
place exposure limits for magnetic fields. So little is known
about magnetobiology, and even whether magnetic fields
can induce long-term effects, that these limits are, to a large
extent, arbitrary.
Additionally, and for esoteric reasons connected to the
geometry of probe coils, small probe coils are inherently
more sensitive than larger coils for a given mass of sample,
so that microprobes which hold only a few microlitres of
sample can give signal-to-noise ratios which rival cryoprobes
(Sakellariou, Le Goff & Jacquinot, 2007). Microprobes are
also useful, of course, when sample volume is limited for
biological reasons, for example in the analysis of the few
microlitres of cerebrospinal fluid in the brains of individual
mice (Griffin et al., 2002).
We noted that nuclei of the same element absorb EM

radiation of a characteristic frequency in a B0 field (Section I).
This is not strictly correct and would make for a poor
analytical technique, as we would only be able to distinguish
different elements within a mixture. We should, more
accurately, have said that nuclei of the same element absorb
EM radiation over a characteristic range of frequencies. This
is demonstrated by the experiment which turned NMR into a
versatile analytical tool: the observation that the 1 H spectrum
of ethanol CH3 CH2 OH consisted of three resonances
(Proctor & Yu, 1950). This was unexpected, because it had
been assumed that all the 1 H nuclei in a sample would absorb
RF radiation of the same energy when moving from the 1/2
to the + 1/2 spin state. Instead, it swiftly became apparent that
the resonance of a nucleus shifted according to its chemical
environment, with ethanols three resonances reflecting its
three 1 H-containing chemical groups; -CH3 , -CH2 - and
-OH (Arnold, Dharmatti & Packard, 1951). The discovery of
this so-called chemical shift, often abbreviated to the Greek
letter , brought NMR into many chemistry departments as
a highly discriminatory analytical probe and this, together
with its penetrance and non-invasiveness, meant that it was
soon adopted in the analysis of biological samples (Odeblad,
Bhar & Lindstrom, 1956).
31 P NMR spectroscopy provides a striking illustration
of how chemical shift may be used in experiments on
muscle function in vivo (Fig. 5A), with 31 P chemical shift
differences readily allowing the spectroscopist to distinguish
resonances from phosphocreatine, inorganic phosphate and
ATP (Fig. 5B). Notice that, in in vivo spectra from muscle
(Fig. 5B), there is no contribution from ADP, which would
produce two resonances in 31 P NMR spectra. This was an
important early finding of biological NMR, demonstrating
that the concentration of ADP measured in assays in vitro
does not reflect the low intracellular free concentrations of
ADP, because ADP binds readily to proteins within the
cell and its effective concentration is reduced accordingly
(Meyer, Brown & Kushmerick, 1985). Moreover, because
the inorganic phosphate resonance is actually an average of
muscle phosphate (H2 PO4 ) and orthophosphate (HPO4 2 ),
it is sensitive to changes in pH, producing a change in
chemical shift which can be used to measure pH changes
during metabolic insults such as ischaemia (lack of blood
flow), hypoxia (lack of oxygen) or cold (Fig. 5B) (Sartoris,
Bock & Portner, 2003).

A
9
older magnets will require the sample to be spun, usually
at around 20 Hz. This spinning averages out any remaining
small B0 field inhomogeneties in the horizontal (often referred
to as xy) plane, improving the final B0 field homogeneity.
(c) Solution-state studies
Fig. 5. The uses of chemical shift: 31 P measurement of muscle

pH in vivo. (A) pH changes play an important role in allowing
animals to adapt to changes in temperature. This has been
investigated in two species of eelpout, a small bony fish,
using 31 P NMR spectroscopy. Sartoris et al. (2003) compared
two species : the temperate Zoarces viviparus (shown in A) and
the cold-adapted Pachycara brachycephalum (Pappenheim, 1912).
(B) Muscle energetics were assessed in both species to measure
intramuscular pH. This is shown for Zoarces viviparus in normal
water (top spectrum) and warm water (bottom spectrum).
Intramuscular pH decreases when the fish is heated. Adapted,
with permission, from (Sartoris et al., 2003).
As 31 P spectroscopy demonstrates, chemical shift is most

useful when signals from nuclei in different chemicals can
be distinguished. This separation may be optimized in four
ways: (a) shimming, (b) sample spinning, (c) using solutionstate studies and (d) magic angle spinning.
(a) Shimming
The EM radiation absorbed and emitted by each nucleus is a
function of both the B0 field and the chemical environment,
so good signal separation requires both of these to be
as homogenous as possible. Because no electromagnet,
regardless of cost, can ever generate a truly homogeneous
B0 field, and because the actual sample itself disturbs the B0
field, the B0 field must be re-homogenized, or shimmed, after
sample introduction; a practice which takes its name from
the engineering trick of using small wedges of material to
adjust the position of a larger object. Shimming used to be a
time-consuming manual process, but is invariably automated
on modern machines, allowing the B0 field homogeneity to
be fine-tuned once the sample is sitting in the magnet.
(b) Sample spinning
When combined with the auto-shimming mechanisms on
modern NMR spectrometers, modern electromagnets can
deliver acceptably homogenous B0 fields. However, many
The need for field homogeneity also explains the usual

use of samples in solution. In a heterogenous sample,
different sample regions will experience different magnetic
environments, most commonly through two phenomena
called magnetic susceptibility and chemical shift anisotropy.
These give rise to local distortions of the B0 field which will,
in turn, produce a smear of energy differences between
two spin states, rather than a sharp line. In solution,
the free tumbling of solutes means that the chemical
environments of those solutes are as similar as possible,
which reduces this line-broadening to give distinct, nonoverlapping resonances. If shimming, spinning and solutions
are effective, resonances will be narrow and well defined,
with a good 1 H linewidththe width of the resonance at
half its heightbeing less than 1 Hz.
We should point out here that the cell is not a true
solid, but rather a semi-viscous liquid, and its metabolite
tumbling rates are fast enough to ensure reasonably narrow
resonances for in vivo tissue studies. Furthermore, many
tissues are homogeneous enough for the effects of chemical
shift anisotropy and magnetic susceptibility to be ignored to
a first approximation. This has allowed clinicians to probe
every organ of the human body by magnetic resonance
imaging and many of these have also been studied by NMR
spectroscopy.
(d) Magic angle spinning
Line-broadening is a particular problem with solid tissue
samples, in which compounds cannot tumble as freely as
they can in solution. A combination of slow relaxation rates
and magnetic susceptibility and chemical shift anisotropy
effects tends to produce a broad amorphorous resonance
in any solid examined by NMR and, at least to a first
approximation, would suggest that in vivo spectroscopy would
be uninformative for following much solid-state biology.
However, resolution of resonances in solid samples can be
improved by the process of magic angle spinning (MAS)
NMR spectroscopy (Lowe, 1959).
Andrew, Bradbury & Eades (1958) observed that many
of the line-broadening effects in solids depended on the
angle at which the sample was placed with respect to the B0
field. They went on to demonstrate that rapidly spinning a
solid sample at the so-called magic angle, 54.7 , resulted in
spectra with significantly reduced linewidths. This is caused
by a combination of physical effects whose explanation is
so complicated that magic really is the most descriptive
adjective! Briefly, various physical effects scale according to
the angle at which the sample is placed with respect to the
B0 field; at the magic angle the angular term becomes zero
and a narrow resonance is produced. Applying this approach
10
to a range of biological tissues, in which most metabolites
are in the semi-viscous cell cytosol, linewidths comparable
to solution-state NMR spectroscopy have been produced in
many different tumour types and neurological tissues taken
either during surgery or post mortem in humans (Cheng et al.,
1997; Griffin et al., 2003b; Millis et al., 1997). This has been
extended further to the spinning of whole organisms, with
mice being technically the most convenient (Wind, Hu &
Rommereim, 2003).
(3) Measuring relaxation
Nuclei in excited spin states usually relax with distinctive
half-lives of around 1001000 ms and, like chemical shift,
changes in the exact value of the half-life can give information
about the environment of the molecules involved. The
relaxation signal is measured as a decay in signal intensity
over a few secondsthe free induction decay (Fig. 3)and
we note in passing that an exactly analogous, although
less informative, phenomenon is seen for light spectroscopy,
in which fluorescence lifetime imaging is used to increase
the information obtainable from visible light excitation of
fluorescent molecules (Suhling, French & Phillips, 2005).
The NMR signal decays through two mechanisms: T1
and T2 . The T1 relaxation time (also called the spinlattice relaxation time) refers to the restoration of the
equilibrium population of the spins along the B0 field
axis (i.e. the superconducting magnet for most NMR
applications) following the RF pulse. This process takes place
largely through nuclear interactions with the surrounding
environment and, because it determines how many nuclei
are available for excitation, affects the rate at which exciting,
B1 , pulses can be applied to a sample during an NMR
experiment. By definition, therefore, the T1 relaxation
rate determines the extent to which a sample may be
saturated during data acquisition: if the T1 relaxation is
long, a long relaxation delay is needed between each pulse
(Section IV.1c).
The T2 relaxation time (also referred to as spin-spin
relaxation time), on the other hand, refers to the loss of
magnetisation coherence that creates the EM radiation
which is emitted during the NMR experiment. This arises
from quantum mechanical effects and the homogeneity of
the magnetic field, and determines how broad the NMR
resonances are. For various reasons, this T2 relaxation takes
place in the xy plane (i.e. perpendicular to the magnet) and
is always faster than T1 relaxation.
(4) Measuring phase
By the late 1950s, NMR spectroscopy had become
established as a useful analytical technique, with its main
disadvantages, then, as now, lying in its low sensitivity
and spatial resolution. This low sensitivity was a particular
problem because spectra were acquired using what was
called continuous wave NMR, in which samples were
excited using a series of single-wavelength RF waves, each
of slightly longer wavelength than the last, in a manner very

similar to modern visible light spectroscopy. Nevertheless,
NMR spectroscopys ability to penetrate deeply into samples
and to identify their composition without damaging them
made it particularly well suited to certain in vivo biological
applications.
If this were all, NMR would be simply another type
of spectroscopy; a useful, but not essential, addition to
modern biologys toolkit. There is, however, a particularly
useful feature of NMR: because RF waves are low energy,
and therefore long wavelength, technical considerations
mean that both their intensities and their phases may be
determined, in contrast to the majority of EM radiation
used by biologists, such as the visible and near-visible light
wavelengths, for which only information about intensities
may be determined. This means that pulse sequences can
be used to simplify spectra and to add informationvisible
light is only slowly being manipulated in this way with the
new generation of ultrafast pulsed lasers (Jonas, 2003; Kroll
et al., 2007).
There are two ways in which NMRs phase information
has been particularly useful. One of the biggest advances in
NMR spectroscopy during the early years of its development
was the fast Fourier transform (Ernst & Anderson, 1966)
which takes a free induction decay in the time domain and
converts it into the frequency domain to produce a spectrum
(Fig. 3). This development significantly speeded up spectroscopy as it allowed the acquisition of a range of frequencies
in a single pulse, rather than necessitating a laborious scan
across a range of frequencies (as occurs in many UV/visible
spectrophotometers). This leaves more time for the biggest
bugbear of most NMR spectroscopiststhe inherent lack of
sensitivity of the approach.
The second application of phase has been the use of pulse
sequence and multidimensional spectroscopy. This will be
dealt with further in Section V.2b.
V. DATA ANALYSIS, OR WHAT CAN THE

FREQUENCY DOMAIN TELL US?
The four parameters of the emitted EM radiation collected
from an NMR experimentintensity, frequency, relaxation
and phase (Fig. 3)contain a great deal of information and
NMR spectroscopy therefore provides a highly quantitative
and discriminatory analytical tool, capable of distinguishing
metabolites in a mixture, or individual amino acids in
a protein. Furthermore, multidimensional outputs can be
generated to show the dependence of one parameter on one,
two or all three of the others, and these inter-relationships
can shed light on a wealth of other information, including
the environment the molecules are found in and whether
they are bound to proteins or to other macromolecules.
Broadly speaking, the information from an NMR
experiment can tell us four things about a sample: (1) how
much of it is present, (2) what it is, (3) where it is and (4) what
it is doing. We will now examine some examples.
11
(1) How much is there? Metabolic foot- and

finger-printing
The simplest, and most common, type of NMR spectroscopy
produces a spectrum of intensity on the y axis against
frequency on the x axisa so-called 1D plot, or frequency
domain (Fig. 3). This is analogous to the intensity versus
wavelength plots generated by other forms of spectroscopy.
Resonances in 1D plots are mathematically multiplied in a
process called line-broadening, which simplifies subsequent
quantification by smoothing the raw data to reduce the
contribution noise makes to the resultant NMR spectrum.
Because the absolute frequency at which nuclei resonate is
primarily a function of the applied B0 field, resonances are
normalized by plotting the x axis as a shift from a reference
standard, so that published chemical shifts are independent of
the magnet strength and so that spectra acquired in different
B0 fields may be compared. Indeed, the use of such a relative
frequency scale is even needed to compare results between
magnets of the same nominal B0 field strength. In order to
ensure that magnets of a given strength do not interfere with
one another if placed in the same facility they will have a
small offset in terms of the magnet field strength. These B0
differences between apparently equivalent magnets are often
greater than proton chemical shifts.
As mentioned in Section IV, chemical shift differences
are usually small, so that if the 1 H nuclei in the external
shift reference, TSP (Section III), resonate at 400 MHz in
a 9.4 T magnet, the C1 1 H nuclei in glucose will resonate
at 400.0021 MHz. Chemical shift is, therefore, given in
parts per million (ppm) so that, in this particular example,
glucose would have a set of C1 1 H resonances centred at
1,000,000 (400.0021 400)/400 = 5.25 ppm. Any given
experiment will cover a certain ppm range, called its
sweep width.
Because NMR is a reproducible quantitative approach,
1D spectra can be used even without identification of
the molecules which have contributed to the spectrum.
As we noted in Section IV.1c, one of the ways in which
signal-to-noise ratios are increased is by performing NMR
spectroscopy in a mode that is termed partially saturated,
where nuclei are excited at a quicker rate than would allow
the NMR signal to decay completely to zero. Fortunately,
saturation factors can be calculated readily, so a simple
linear relationship exists between the area under a partially
saturated peak and the concentration of the nuclei that
produce the signal. This gives us a characteristic metabolic
phenotype from every sampleakin to a barcode, or a
snapshotwhich can be statistically separated from another
set of samples using multivariate analysis such as principal
components analysis, even if the individual resonances
cannot be identified (Bundy et al., 2007; Raamsdonk
et al., 2001).
This snapshot approach has proved itself to be particularly
useful in functional genomic studies, in which genes are
altered and the phenotype of the resulting mutants is
examined to provide clues about gene function. Phenotypes
have traditionally been scored using a very limited range
of parameters, such as growth rate in yeast. However,

changes in gene activity will often result in apparently
silent mutations, as other regulatory processes compensate
to produce no discernible change in gross phenotype
(Teusink et al., 1998). In such cases, a metabolic snapshot,
quantifying resonances without needing to identify them,
may be used as a multiparametric and extremely sensitive
phenotype. If the metabolic snapshot comes from cell,
tissue or organism preparations, this approach is known as
metabolic fingerprinting; if the snapshot looks at cell media
or excretory products, it is known as metabolic footprinting
(Allen et al., 2003).
Both finger- and footprinting work best with sensitive
and abundant nuclei, such as 1 H; one of the most famous
examples of this approach is the highly cited functional
analysis by co-responses in yeast (FANCY) manuscript
by Raamsdonk et al., 2001. This work showed that a
number of Saccharomyces cerevisae (yeast) strains with similar
growth rates had markedly different 1 H NMR spectra
which could be used to distinguish glycolytic mutants
from oxidative phosphorylation mutants. FANCY has
recently been extended to identify metabolic modules
in S. cerevisiae (Bundy et al., 2007). Briefly, extracellular
metabolite profiles, metabolic footprints, were quantified
by 1 H NMR spectroscopy and analyzed using principal
components analysis (PCA) (Fig. 6). This is an unsupervised
statistical analysis technique that fits a large multivariate
dataset onto a multidimensional set of orthogonal axes. These
axes, which are called principal components, are chosen so
that each axis explains as much of the variance remaining in
the dataset as possible. The overall aim of PCA is to reduce
the number of parameters needed to describe the differences
among datasets and, properly applied, PCA is a powerful
way to classify mutants and their associated genes (Fig. 6).
As well as the convenience of not having to identify
metabolites, snapshot approaches can also be used in high
throughput screens where metabolite identification can be
postponed until an effect has been observed. Using such
an approach, the consortium for metabonomic toxicology
(COMET) investigated around 150 model liver and kidney
toxins during a three-year study using NMR-based analysis
of urinary metabolites (Lindon et al., 2005; 2003). It is
hoped that such an approach will allow the generation
of expert systems where liver and kidney toxicity can be
predicted for model drug compounds, with the databases
being easily transferable among laboratories. In addition the
approach has also been used to screen human populations
to understand disease processes (Brindle, 2002; Kirschenlohr
et al., 2006) and monitor the effects of diet (Lenz et al., 2004).
This metabolomic approach to analyzing gene function
has three advantages over gene- or microarray-based
approaches. First, it measures functional entities, which
reduces biological noise. Second, there are a smaller number
of metabolites than there are genes. Which simplifies analysis:
at the last count, around 1500 metabolites were found in
the yeast S. cerevisiae as opposed to 6000 genes (Goffeau et al.,
1996; Herrgard et al., 2008). Indeed, the components of
12
0.6
PUT3
Glucose
PUT2
HO
0.4
0.5
PUT1
URA3
PC1 loadings
0.2
PC2
1.0
URA8
URA5
0.0
URA7
-0.2
0.0
-0.5
PPR1
-0.4
URA2
URA10
-0.6
-1.0
-1.0 -0.5
0.0
0.5
1.0
1.5
2.0
2.5
PC1
Variable order
Fig. 6. Metabolic footprinting of yeast mutants. (A) principal component 1 (PC1) (x axis) versus principal component 2 (PC2) (y axis)
scores plotted for 1 H nuclear magnetic resonance (NMR) footprints from eleven Saccharomyces cerevisiae yeast strains: three proline
utilization mutants (PUT1, PUT2, and PUT3; red), seven pyrimidine biosynthesis mutants (URA2, URA3, URA5, URA7, URA8,
URA10, PPR1; blue), and one control (HO, black). The centre of each ellipse is the mean of the principal component on that axis,
with the margins indicating one standard deviation. The principal components of most strains cluster together, but those of URA7
and URA8 sit well away from the others, on the right of the plot. (B) The difference between URA7 and control spectra is plotted
as a loadings plot, with the variable order (the pattern recognition softwares term for chemical shift) on the x axis. Loadings plots
highlight which metabolites are most discriminatory in the PCA plot. In this example, the regions containing glucose are all shown
to have increased in the loading for PC1, demonstrating that URA7 has increased glucose concentrations relative to the control
strain. Taken together, these results suggest that the URA7 and URA8 gene products are subject to different regulatory controls
than the other genes. Adapted, with permission, from Bundy et al., (2007).
central metabolism which dominate the metabolome only

number a hundred or so metabolites. Third, it is contextdependent because it can measure how the plasticity of a
mutant phenotype in response to environmental and external
stimuli.
When considering large-scale analyses of biological mixtures, the main alternative to NMR spectroscopy has
traditionally been mass spectrometry. Mass spectrometry
is undoubtedly the more sensitive technique and in tissue
extracts in which 3060 metabolites might be detected by
NMR, a relatively cheap GC-MS instrument will often find
more than 100 (Atherton et al., 2006), with the upper limit
to metabolite numbers thought to depend on the constraints
of cellular osmolarity (Krishnan, Kruger & Ratcliffe, 2005).
However, despite recent improvements in mass spectrometry sensitivity and robustness GC-MS still identifies less
than 20% of the metabolites present, and methods such
as liquid-chromatography-based MS and Fourier transform
MS, as well as niche techniques such as Coulombic arrays,
fare little better. The fact that these techniques can still measure only a small proportion of the total metabolome (see
Ellis et al., 2007) for a broad review of metabolomic
techniques suggests that there are problems associated with
metabolite extraction and resolution, rather than technique
sensitivity.
In any case, because NMR analyses are reproducible, can
detect certain critical metabolites which define the regulatory
metabolic networks (Delgado et al., 2004) and, crucially,
may be used on a sample before derivitization for GC-MS,
there is increasing consensus that NMR spectroscopy and
mass spectrometry are best used in conjunction, rather

than in competition, to maximize the coverage of the
metabolome.
(2) What is it? Metabolic profiling and protein NMR
spectroscopy
Metabolic footprinting assigns a quantity to each peak, but
1D spectra of the frequency domain also carry qualitative
information about a sample. We noted in Section IV.2 that
the spectrum of ethanol gave three distinct resonances and,
in general, each chemical will have its own characteristic
pattern of peaks. There are a number of ways in which
we can identify these resonances, drawn from analytical
chemistry. These are: (a) chemical shift, (b) multidimensional
NMR, and (c) hyphenated NMR.
(a) Chemical shift and spin-spin coupling
As we noted in Section IV.2, the energy difference between
spin states is dependent upon nuclear environment, so
excited nuclei from one element, but in different nuclear
environments, will emit slightly different RF wavelengths
of EM radiation. In NMR, the energy of the emitted
EM radiation is expressed as the chemical shift, , and
the exact value of gives a reasonable indication of the
molecules identity.
This identification may be narrowed down still further
if we consider each resonances splitting pattern. Many
resonances at a given chemical shift will be split into two,
13
three, or more sub-peaks, called, respectively, doublets,

triplets and multiplets. These splittings arise through a
phenomenon called spin-spin coupling; each nucleus behaves
as a tiny magnet within the molecule, so that nucleus A will
affect the energy of nucleus B, and vice versa, so long as
they are positioned close to one another. Because each
molecule has a characteristic number and pattern of these
sub-peaks, a simple comparison of unidentified peaks with
standard reference spectra will often allow identification
of the metabolites in a sample. Accordingly, a number
of NMR spectral databases have been set up to aid this
metabolite assignation; these databases include the Human
Metabolome Database (Wishart et al., 2007) and the Madison
Metabolomics Consortium Database (Cui et al., 2008), as well
as a number of proprietary software tools.
can use RF pulse sequences to add information to a spectrum

which might help with assignation.
For example, one of the most efficient methods for
determining which compounds are in a mixture is to use
heteronuclear pulse sequences which detect which 1 H nuclei
are connected to which 13 C nuclei. A variety of such pulse
sequences exist, which have unfortunately opaque names
like heteronuclear single quantum coherence (HSQC), heteronuclear multiple quantum coherence (HMQC) and
heteronuclear multiple bond coherence (HMBC) (Braun,
Kalinowski & Berger, 1998). These 1 H-13 C heteronuclear
pulse sequences are especially useful because they combine
the relatively high sensitivity of 1 H NMR spectroscopy with
the increased dispersion of 13 C NMR spectroscopy, the latter having a chemical shift range of approximately 250 ppm
compared with approximately 15 ppm in 1 H NMR spectroscopy. Thus by detecting a combination of 1 H and 13 C
resonances in a metabolite, we can rapidly narrow down
the identity of the compounds that could be present in that
mixture.
For instance, in order to understand tissue energetics,
it is important to know the relative levels of highenergy phosphates. The sperm of many animals contain
high levels of ATP, but mammalian sperm do not,
containing instead phosphodiesters and phosphomonoesters.
Because phosphomonoester resonances are poorly disperse,
multidimensional heteronuclear NMR spectroscopy has
been used to identify the exact species of phosphomonoester
present and to further our understanding of mammalian
fertilization and reproduction (Fig. 7).
(b) Multidimensional NMR

The previous section showed that, in theory, unknown
compounds in a spectrum can be identified by their
chemical shift and by the number and pattern of their
sub-peaks in the frequency domain. Unfortunately, because
the differences in chemical shift are relatively small for 1 Hcontaining molecules, biological 1 H NMR spectra are often
cluttered with many overlapping resonances that make peak
identification difficult: we say that 1 H peaks are poorly
dispersed. This dispersion problem occurs in most forms of
spectroscopy, but highlights one of the strengths of NMR;
because it is possible to shape RF waves and measure their
phase, and because it is also possible to detect the nuclei of
different elements simultaneously in a single experiment, we
A
Fig. 7. Pulse sequences in multidimensional nuclear magnetic resonance (NMR) spectroscopy. (A) Two-dimensional (2D) 1 H, 31 P
heteronuclear multiple bond correlation (HMBC) NMR spectroscopy of lyophilized boar sperm extracts. HMBC is a pulse sequence
which detects 1 H nuclei separated by a given number of bonds from 31 P nuclei and, in this case, shows a chemical shift correlation
between the 31 P phosphomonoester peak (PME) and a given 1 H peak at around 4 ppm. The inset shows an expansion for the PME
cross-peak ; glycerol 3-phosphorylcholine (GPC) and inorganic phosphate (Pi ) peaks are also labelled on the 31 P spectrum. (B) 2D
1 H, 13 C gradient-selected heteronuclear single quantum coherence (HSQC) of boar sperm extracts. HSQC is a pulse sequence
which observes 1 H nuclei attached to 13 C nuclei and this spectrum shows that the 1 H peak identified in A is linked to an adenine
base, which identifies the major phosphomonoester component as AMP. Adapted, with permission, from Kalic et al., (1997).
14
The field where multi-dimensional NMR spectroscopy has
most impressively been used is its use in the determination
of protein structures as first suggested by among others Kurt
Wuthrich (Wuthrich, 1990). Proteins may have thousands
of NMR resonances associated with them, and thus simple
one-dimensional 1 H NMR spectra are highly congested. To
circumvent this dispersion problem, spectra from proteins are
usually collected across multiple dimensions often associated
with different nuclei. In particular, because the backbone
of peptides consists of amide linkages, the use of 1 H, 13 C
and 15 N multi-dimensional NMR spectra is common in
protein NMR spectroscopy. These experiments can consist
of two-dimensional NMR spectra such as 1H-13 C HSQC and
1 H-15 N HSQC experiments which take hours to 23 days
to acquire depending on the protein, whether it has been
artificially labelled with amino acids containing 13 C and 15 N
and the field strength of the magnet used. Alternatively,
more time-consuming three-dimensional experiments can
be performed that simultaneously measure the interactions
between 13 C, 15 N and 1 H or experiments. Furthermore,
there are certain NMR approaches that do not rely on
magnetisation being carried through bonds as occurs in the
HSQC experiment but move through space. The nuclear
Overhauser effect spectroscopy (NOESY) pulse sequence is
used to measure how close atoms are in proteins and thus
useful in placing distance constraints on the size and shape
of a protein. The field of protein spectroscopy is a large and
complex one and we direct the interested reader to specialist
reviews in this area, notably Wuthrich (1990), Reid et al.,
(1997) and Shin, Lee & Lee (2008).
(c) Hyphenated NMR
Multidimensional NMR adds another dimension (or
dimensions) to a spectrum and effectively adds more
information to aid peak assignation. This extra dimension
does not have to be an NMR one and it is often desirable
to combine NMR with other techniques. For example,
we can include online chromatographic separation, which
fractionates the sample before running a sequence of
NMR spectra. This is analogous to GC-MS and liquidchromatography mass spectrometry (LC-MS). In addition to
separating metabolites according to their chromatographic
properties, liquid chromatography also concentrates
metabolites into chromatographic peaks. By increasing the
local concentrations of metabolites, the sensitivity of the
NMR experiment can be increased (Bailey et al., 2002).
(3) Where is it? Metabolite environment
Differences in chemical shift reflect differences in the
environment of a nucleus. So far, we have assumed that
different environments mean similar nuclei in different
molecules. However, they may also reflect the same
molecules in different environments. A number of properties
of the spectral peak may be affected by environment. Most
simply, the chemical shift may change, as is the case with
the inorganic phosphate peak in 31 P spectroscopy, whose

chemical shift is pH dependent (Section IV.2 and Fig. 5).
However, the relaxation times T1 and T2 (Section IV.3) and
the diffusion properties (which can be measured by NMR)
can also be used to provide information about molecular
environments.
For relaxation times, both T1 and T2 are influenced
by how fast individual metabolites tumble in the B0
field. Metabolites in a restricted environment, and which
cannot tumble freely, produce broad resonances which
are most easily distinguished by direct measurement of
T1 and T2 relaxation times. This approach has been
used to demonstrate the restrictive environment of the
mitochondrion for both proteins (Haggie & Brindle, 1999)
and metabolites (Bollard et al., 2003). In addition, so-called
relaxation editing has been used to distinguish different
metabolites in complex biofluids such as plasma using pulse
sequences (Liu, Nicholson & Lindon, 1996).
An alternative to relaxation-based approaches is to
distinguish metabolites by their diffusion properties. This
approach uses a magnetic gradient in which the B0 field
varies, usually linearly, across the sample. A second B0
gradient is then applied that is equal in magnitude but
opposite in sign. For metabolites that do not move rapidly
these two magnetic gradients will cancel out exactly.
However, the nuclei of rapidly moving metabolites will
receive a net magnetisation which will vary according to
the position of the nuclei. Averaging over the whole sample,
this results in a broadening of the resonance. This approach
has also been used to distinguish low molecular weight
metabolites from lipoproteins and bound metabolites in
blood plasma and biological tissues such as intact tumours
using so-called diffusion weight spectroscopy (Liu et al., 1996;
Griffin et al., 2003a).
(4) What is it doing? Metabolite kinetics/behaviour
As with any form of spectroscopy, NMR can follow changes
in peak intensity over time to give an idea of the timecourse
of metabolite reactions in a way which is not too different to
the use of radiolabels or similar metabolite tags. However,
NMR is once again set above other forms of spectroscopy by
our ability to measure the phase of EM radiation and thus
to use pulse sequences. In particular, using an experiment
referred to as magnetization transfer, NMR spectroscopy
may, effectively, introduce a metabolite label at any point
during the course of an experiment (Bittl & Ingwall, 1985;
Degani et al., 1985). For example, magnetization transfer has
been used to examine the transfer of phosphate between
phosphocreatine and ADP (Brindle & Radda, 1985). Here,
the phosphocreatine phosphorus nucleus is irradiated by
RF waves in such a manner that its population of spins is
cancelled out (and hence the resonance would be invisible in
the NMR effect). When this phosphate group is transferred
from phosphocreatine to ADP to produce ATP, it carries
its magnetisation with it. This shows up as a reduction in
the signal of the ATP phosphate. The rate of reduction
in the intensity of the ATP phosphate resonance can then
be used to estimate the activity of the creatine kinase which
15
catalyses the conversion of phosphocreatine to ATP (Brindle

& Radda, 1985).
With the development of new protein labeling schemes,
NMR spectroscopy can now reveal structural and binding
details of large protein complexes. Binding of substrates will
induce changes in the structure of the enzyme and this in
turn will alter the chemical shift of the resonances observed in
that protein, especially of those involved in the binding site.
For instance, Velyvis et al., (2007) studied ligand binding to
the 306 kDa aspartate transcarbamoylase (ATCase) enzyme
using highly deuterated, 1 H,13 C-methyl labelled ATCase
together with methyl-transverse relaxation optimized NMR
spectroscopy. Previous research had demonstrated that
the cooperative binding of active site ligands follows
the Monod-Wyman-Changeux (MWC) model of allostery,
which proposes the existence of two conformations of an
enzyme in equilibrium, T and R. The T form has a lower
affinity for substrates, and as more sites in the enzyme bind
substrate, the equilibrium shifts toward the R state. The
authors established that for ATCase, which catalyzes the
first step in pyrimidine biosynthesis, only the T state of the
enzyme can be observed in NMR spectra of unliganded
ATCase. However, the binding of nucleotides such as ATP
or CTP shifts the T-R equilibrium so that correlations from
the R state become visible, consistent with the MWC model,
with these resonances shown in black in Fig. 8. This study
emphasizes the utility of modern solution NMR spectroscopy
in understanding protein function, even for systems with high
molecular masses (Velyvis et al., 2007).
Finally, in just the same way that 14 C-labelled compounds have been used to determine substrate metabolism in
perfused tissues and the whole organism, 13 C-labelled compounds can be used and detected by 13 C NMR spectroscopy.
Although 13 C NMR spectroscopy is of inherently low sensitivity when compared to 1 H NMR spectroscopy 13 Cs
low gyromagnetic ratio making it an order of magnitude less
detectable than 1 Hit can be performed in vivo or in situ in
real time allowing the measurement of the rates of processes
such as the tricarboxylic acid (TCA) cycle (Malloy, Sherry
& Jeffrey, 1990), or even mitochondrial transport (Yu et al.,
1996). Furthermore, unlike radioactivity-based approaches,
one can also determine the exact position of the label,
which has allowed the study of metabolic compartmentation
within the brain (Badar-Goffer et al., 1990; Griffin et al., 1998)
and substrate selectivity within the heart (Malloy, Sherry &
Jeffrey, 1988).
Moreover, the problem of poor sensitivity is now being
addressed for some applications involving 13 C NMR
spectroscopy, thanks to a resurgence of interest in a technique
called hyperpolarization (Golman et al., 2003; Schroeder
et al., 2008), in which huge population differences can be
built up between ground and excited spin states in certain
molecules. In hyperpolarization, the sample is mixed with
a polarization transfer agent before being frozen solid. The
polarization transfer agent is typically something with an
unpaired electron which can be excited by microwave
radiation. This, incidentally, is electron spin resonance,
Fig. 8. Protein spectroscopy. Two-dimensional (2D) 1 H-13 C

methyl-transverse relaxation optimized spectroscopy (TROSY)
heteronuclear multiple quantum correlation (HMQC) spectrum
of U-[2 H] Ile-[113 CH3 ]-labelled aspartate transcarbamoylase
(ATCase) recorded at 800 MHz, showing 27 resonances which
correspond to the 27 Ile residues in ATCase. TROSY is a pulse
sequence that specializes in the analysis of large proteins. For
normal one-dimensional (1D) and 2D spectroscopy, resonance
linewidths increase with molecular mass, producing short T2
relaxation times, broad resonances and correspondingly poor
resolution. TROSY avoids this line-broadening and produces
narrow peaks for large proteins, allowing the discrimination of
resonances not possible using other pulse sequences. By way
of demonstration, the arrows point to the cross-peaks through
which the four 1D traces have been drawn. These four traces
show the high signal-to-noise ratio and illustrate the sensitivity
of the spectrum, which comes from solution studies and the very
high B0 field strength (800 MHz). Peaks from the r and c chains
of ATCase are colored red and black, respectively, with the
change in structure induced by ligand binding. Adapted, with
permission from (Velyvis et al., 2007).
which is analogous to NMR except that it occurs at a higher

EM frequency, since electrons have larger energy differences
between their two spin states. In the solid, frozen, state
magnetization is transferred from the polarisation transfer
agent onto the sample. The sample is then rapidly thawed
before being used for conventional NMR spectroscopy.
Hyperpolarization can build up huge population differences,
but with the disadvantage that differences begin to disappear
through T1 relaxation as soon as the sample is thawed
(Golman et al., 2003). As a result most applications have so
far involved carbonyl 13 C resonances, as these tend to have
relatively long T1 times (tens of seconds) and thus allow
the magnetization to be maintained during the experiment.
Despite this limitation, this approach has been used to
follow metabolism in tumours and heart tissue with exquisite
temporal resolution (Fig. 9).
VI. CONCLUSIONS
(1) NMR spectroscopy is a powerful and flexible technique
which allows biologists to study the molecular and
16
(7) The frequencies, or chemical shifts, of peaks in

an NMR spectrum can be used to identify which
compounds are present in a mixture.
(8) The half-lives, or relaxation times, of peaks in an
NMR spectrum can give information about molecular
environments.
(9) NMR spectroscopy has more analytical powerand is
more complicatedthan other forms of spectroscopy
because we are able to measure the phase of RF
radiation. This allows us to study the connectivities
between magnetic nuclei, giving a tool with which to
study protein structure and dynamics.
(10) We have focussed mainly on small-molecule
spectroscopic applications, despite huge literatures
on magnetic resonance imaging and protein NMR
spectroscopy. However, we hope that interested
readers will now be armed with the key concepts
of NMR spectroscopy and will be in a position to
benefit from more specialised reviews and primary
publications.
Fig. 9. Hyperpolarization. (A) An example time course of

spectra acquired from a rat heart in vivo over 60 s of
data acquisition. The arrival of [1-13 C]pyruvate can be
seen, followed by its subsequent decay back to thermal
equilibrium levels. The appearance of the metabolic products,
[1-13 C]lactate, [1-13 C]alanine and bicarbonate (H13 CO3 )
follows shortly afterwards through [1-13 C]pyruvate metabolism.
(B) An example of the temporal variation in the fitted peak areas
of each metabolite over the 60 s time course. The pyruvate area
has been reduced by a factor of 10 to improve visualization.
This figure was supplied to the authors by Damian Tyler at the
University of Oxford.
(2)
(3)
(4)
(5)
(6)
cellular environments of magnetic nuclei. Commonly

studied magnetic nuclei include 1 H, 13 C and 31 P.
In NMR spectroscopy experiments, magnetic nuclei
are placed in a strong B0 electromagnetic field and
excited from lower energy spin states to higher energy
spin states by the absorption of EM radiation generated
by a second, B1 , electromagnetic field. These excited
nuclei then relax by emitting EM radiation of slightly
less energy; both exciting and emitted radiation are in
the RF range.
Because NMR spectroscopy uses low-energy RF
radiation which is absorbed by nuclei, it can penetrate
deeply into tissue and is non-invasive.
Unfortunately, the low energy of RF radiation also
makes NMR spectroscopy a relatively insensitive
technique. There are a number of practical
ways around this low-sensitivity problem, including
hyperpolarization.
The emitted RF radiation has four parameters which
may be analyzed for information about the sample:
intensity, frequency, half-life and phase.
The intensities of peaks in an NMR spectrum reflect
molecular concentrations, so NMR spectroscopy
is often used as a standard, automated, highthroughput analytical technique, especially suitable
for metabolomics.
VII. ACKNOWLEDGEMENTS
J.H.B. is funded by a Leverhulme Early Career Fellowship.
Additionally, we are grateful to the BBSRC, MRC, NERC
and Royal Society for financial support. We would like to
thank Reza Salek, Duncan MacInnis and Juan Castrillo at
the University of Cambridge for the yeast HSQC spectrum
in Fig. 2, Oliver Jones at the University of Cambridge for
the rice spectra in Fig. 4, Franz-Josef Sartoris at the AlfredWegener-Institut for the eelpout spectra in Fig. 5, Kevin
Brindle at the University of Cambridge for the yeast spectra in
Fig. 6, Gunter Kamp at Johannes Gutenberg University for
the sperm spectra in Fig. 7, Lewis Kay and Algirdas Velyvis
at the University of Toronto for the ATCase spectrum in
Fig. 8 and Damian Tyler at the University of Oxford for the
spectra in Fig. 9.
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NMR Introduction To Biological Sample

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Biol. Rev. (2010), pp. 000000.

An introduction to biological nuclear magnetic

(Received 26 April 2009; revised 30 July 2010; accepted 10 August 2010)

John H. F. Bothwell and Julian L. Griffin

(3) Measuring relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

considered to arise from the spinning motion of its electrical

Biological NMR spectroscopy

Fig. 1. Some physical properties of the elctromagnetic

(Jacobson, Anderson & Arnold, 1954). Only three years

II. RUNNING AN NMR EXPERIMENT

John H. F. Bothwell and Julian L. Griffin

Fig. 2. Sample one-dimensional (1D) and two-dimensional

and Fig. 2). The emitted RF radiation has four parameters

III. SAMPLE PREPARATION

nucleus makes up only a very small part of any atom, RF

Biological NMR spectroscopy

clouds which make up the bulk of an atoms volume, so UV

Fig. 4. Different nuclear magnetic resonance spectra from

John H. F. Bothwell and Julian L. Griffin

Biological NMR spectroscopy

ratio for a given mass of sample: (a) they increase B0 ,

and hence why absorption peaks are commonly known as

John H. F. Bothwell and Julian L. Griffin

(d) Temperature reduction

We noted that nuclei of the same element absorb EM

Biological NMR spectroscopy

(c) Solution-state studies

Fig. 5. The uses of chemical shift: 31 P measurement of muscle

As 31 P spectroscopy demonstrates, chemical shift is most

The need for field homogeneity also explains the usual

John H. F. Bothwell and Julian L. Griffin

V. DATA ANALYSIS, OR WHAT CAN THE

Biological NMR spectroscopy

(1) How much is there? Metabolic foot- and

of parameters, such as growth rate in yeast. However,

John H. F. Bothwell and Julian L. Griffin

central metabolism which dominate the metabolome only

mass spectrometry are best used in conjunction, rather

Biological NMR spectroscopy

three, or more sub-peaks, called, respectively, doublets,

can use RF pulse sequences to add information to a spectrum

(b) Multidimensional NMR

John H. F. Bothwell and Julian L. Griffin

Biological NMR spectroscopy

catalyses the conversion of phosphocreatine to ATP (Brindle

Fig. 8. Protein spectroscopy. Two-dimensional (2D) 1 H-13 C

which is analogous to NMR except that it occurs at a higher

John H. F. Bothwell and Julian L. Griffin

(7) The frequencies, or chemical shifts, of peaks in

Fig. 9. Hyperpolarization. (A) An example time course of

cellular environments of magnetic nuclei. Commonly

Biological NMR spectroscopy

Beckwith-Hall, B. M., Nicholson, J. K., Nicholls, A. W., Foxall, P. J. D.,

Griffin, J. L., Nicholls, A. W., Keun, H. C., Mortishire-Smith, R. J.,

John H. F. Bothwell and Julian L. Griffin

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