An introduction to biological nuclear magnetic resonance ...reilly/NMR/article1.pdf · Biol. Rev. (2010), pp. 000–000. 1 doi: 10.1111/j.1469-185X.2010.00157.x An introduction to

Biol. Rev. (2010), pp. 000–000. 1doi: 10.1111/j.1469-185X.2010.00157.x

An introduction to biological nuclear magneticresonance spectroscopy

John H. F. Bothwell1,2,3∗ and Julian L. Griffin4

1 Medical Biology Centre, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK2 Marine Biological Association of the UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK3 UMR 7139, Station Biologique, Place Georges Teissier, 29682, Roscoff Cedex, France4 The Hopkins Building, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK

(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 inacquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, ratherthan imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which toaddress a number of biological problems. Using detailed examples, we discuss the use of 1H NMR spectroscopy inmixture analysis and metabolomics, the use of 13C NMR spectroscopy in tracking isotopomers and determining theflux through metabolic pathways (‘fluxomics’) and the use of 31P NMR spectroscopy in monitoring ATP generationand intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probethe physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and howit 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 recentadvances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—areopening new avenues for today’s biological NMR spectroscopists.

Key words: 13C, 1H, hyperpolarization, magic angle spinning, magnetization transfer, metabolomics, multidimensionalNMR, NMR spectroscopy, 31P, pulse sequences.

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2II. Running an NMR experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

III. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4IV. Data acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

(1) Measuring intensity and optimizing the signal-to-noise ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6(a) Increasing B0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7(b) Signal averaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7(c) Increasing the population difference in a sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7(d) Temperature reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8(e) Volume reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

(2) Measuring energy—chemical shift, or δ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8(a) Shimming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9(b) Sample spinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9(c) Solution-state studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9(d) Magic angle spinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

* Address for correspondence: (Tel: +44 (0)28 90972269; Fax: +44 (0)28 90975877; E-mail: [email protected] or [email protected])

Biological Reviews (2010) 000–000 © 2010 The Authors. Biological Reviews © 2010 Cambridge Philosophical Society

2 John H. F. Bothwell and Julian L. Griffin

(3) Measuring relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10(4) Measuring phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

V. Data analysis, or what can the frequency domain tell us? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10(1) How much is there? Metabolic foot- and finger-printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11(2) What is it? Metabolic profiling and protein NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

(a) Chemical shift and spin-spin coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12(b) Multidimensional NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13(c) Hyphenated NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

(3) Where is it? Metabolite environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14(4) What is it doing? Metabolite kinetics/behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15VII. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

I. INTRODUCTION

Nuclear magnetic resonance, or NMR, spectroscopy usesradiofrequency waves to reveal information about magneticnuclei. Since the word ‘spectroscopy’ describes any techniquein which electromagnetic (EM) radiation is used to probeatoms (Figure 1), NMR spectroscopy is only one of a rangeof spectroscopic methods in everyday biological use.

However, for reasons which we will consider herein, NMRdiffers from other forms of spectroscopy in three importantways. First, NMR looks at how the nuclei of a specific,user-selected, chemical element are distributed amongst themolecules of a sample, giving NMR a broader range oftargets than most spectroscopic techniques. Second, NMRsignals are sensitive to the local surroundings of the nucleiunder observation, providing a tool that can probe thechemical and physical environment of an atom and whichcan reveal more information about a given sample thanmost other spectroscopic techniques. Third, NMR is morehighly penetrating—but, usefully, less damaging—thanother forms of spectroscopy. Why is NMR spectroscopyso powerful?

To answer this question, imagine standing at the Earth’sNorth Magnetic Pole—currently an ice floe in the CanadianArctic—with all of the World’s compasses pointing towardsus. Their needles can, with a little effort, be forced to pointin another direction, but are in their most stable states whenaligned with the Earth’s magnetic field and will relax backto pointing north as soon as they are released. A numberof common biological elements have nuclei which behavemuch like these compasses; they will align themselves in amagnetic field, may be forced to point in another direction,and will relax back to point ‘north’ once released, usuallywithin a few hundred milliseconds.

There are two things which we need to add to thismagnetic compass analogy to understand enough NMRfor most biological applications. First, a magnetic compasscan be deflected to point in any direction, but quantummechanical laws restrict magnetic nuclei to pointing in amuch more limited set of directions. These ‘directions’ areactually nuclear energy levels called ‘spin states’ where ‘spin’refers to the fact that the magnetism of a given nucleus may be

considered to arise from the spinning motion of its electricalcharge. For instance, when the 1H and the 13C nuclei are ina magnetic field, they each have only two allowed spin states,which are called +1/2 (‘spin up’) and −1/2 (‘spin down’). 1H and13C are therefore known as ‘spin 1/2 nuclei’. It is importantto realize that, in any population of 1H and 13C nuclei, notall of the nuclei will be in the same spin state; some will bein the higher energy, excited, +1/2 state, the rest will be inthe lower energy, ‘ground’, −1/2 state, with their proportionsdepending on the energy difference between the two spinstates—the higher the energy difference, the more nuclei willbe in the −1/2 state. Other nuclei, known as ‘quadrupolar’nuclei snd including the biologically informative 2H, 18Oand 23Na, have spins >1/2 and will also have detectableNMR 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 soinwill give a detectable NMR signal.

The second point which we need to add to ourcompass analogy is that individual magnetic nuclei maybe moved, or ‘flipped’, from one spin state to anotherby radiofrequency (RF) waves whose exact frequencies arediagnostic for the chemical element involved. This RF-wave-induced flipping of magnetic nuclei was first observed over70 years ago by Isidor Rabi’s group at Columbia University(Rabi, 1937; Rabi et al., 1938) and named nuclear induction.However, because some of the early theories to describe thisnuclear flipping employed the idea of nuclei resonating at thefrequencies at which they absorbed RF waves, Rabi’s ‘nuclearinduction’ quickly became known as nuclear magneticresonance, or NMR (Gorter & Broer, 1942). We will referto NMR spectroscopy throughout this review, although itshould be noted that, in a clinical context, it is commonpractice to drop the n-for-nuclear to avoid troubling patientsunnecessarily with unfounded fears of nuclear radioactivity.Under clinical conditions, therefore, NMR spectroscopy isusually shortened to magnetic resonance spectroscopy, orsimply MRS.

Following Rabi’s initial physics-based discoveries, NMR’sbiological applications began to be realized in 1954,when a group at Stanford used 1H NMR spectroscopyto show that DNA strands have large hydration shells


Biological NMR spectroscopy 3

A

B

Fig. 1. Some physical properties of the elctromagneticspectrum. (A) Electromagnetic (EM) radiation is packaged intophotons. Each photon has an associated frequency, ν, andwavelength, λ. Matter can only absorb EM radiation at discretewavelengths, because the energy, E, of any beam of EMradiation is related to its wavelength, λ, by a constant, h, inthe formula E = h/λ. (B) Different regions of the EM spectrumare used for different types of spectroscopy. UV, ultraviolet;Vis visible; IR, infrared; RF, radiofrequency; NMR, nuclearmagnetic resonance.

(Jacobson, Anderson & Arnold, 1954). Only three yearslater, biomolecular NMR had advanced to the stage wherean entire intact protein, ribonuclease, was being examined(Saunders, Wishnia & Kirkwood, 1957) and, today, NMRspectroscopy has become one of the most powerful andflexible analytical techniques available to biologists, beingone of the few methods which give analytical informationfrom deep within living tissue. The solid theoreticalfoundations of the effect have been exploited in innovativetechniques 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 increasinglydriving post-genomic functional studies (Bundy et al., 2007).

This makes it a regrettable, and preventable, shamethat many biologists still view NMR as a prohibitivelymathematical technique. Not only is NMR groundedin a phenomenon which most biologists use withoutdemur in other contexts—spectroscopy using EM radiation(Fig. 1B)—but many of the applications of NMR can beunderstood in non-mathematical terms. Indeed, modernNMR spectrometers allow their users to acquire andanalyze complex spectra without needing any deep physicalunderstanding of the concepts by which the data aregenerated.

For these reasons, and because of the ever-increasingworkload which modern ‘-omics’ and structural biologyplace on high-throughput analytical techniques, we havewritten this review for the large professional and student

audience which is interested in the biological applicationsand potential of NMR spectroscopy, but which lacks themathematical background to tackle traditional theoreticalapproaches to the subject. We will start by looking at theroutines involved in running a typical NMR experiment, wewill then explain how these routines reflect the physical basisof NMR and we will end by demonstrating what informationthese routines can give us.

This review has three main sections—the first two(Sections III and IV) are a brief introduction to the principlesbehind running an NMR experiment, the third (Section V)is a more detailed look at the information which canbe obtained from NMR spectroscopy. For those whowish 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.

II. RUNNING AN NMR EXPERIMENT

NMR experiments are, in practical terms, fairly straightfor-ward. For simplicity’s sake, we will break them down intothree parts—sample preparation, data acquisition and dataanalysis—each of which will be covered in separate sectionsof this review.

During sample preparation (Section III), the tissue ofinterest is prepared, most commonly as a solution in anarrow-walled glass tube, and put into a high-field electro-magnet whose large coil generates a strong B0 –pronounced‘B nought’—field (Section IV). Once inside the B0 magnet,the sample sits in a second, smaller, electromagnetic coilwhich is found at one end of a poster-tube-sized cylindricalprobe. This second coil is, understandably, known as theprobe coil (Section IV).

During data acquisition (Section IV), the sample—stillin the magnet’s B0 field—is irradiated with RF wavesgenerated by the probe coil. RF waves have electrical andmagnetic components, so these irradiating RF waves areoften known by their ‘magnetic’ name, which is the B1 field.These RF waves—sometimes grouped together in a clustercalled a pulse sequence (Sections IV.1.c and V.2.b)—flipnuclei in the sample from one spin state to another. Themore power applied in the RF wave, the more nucleiare flipped, a process known as saturation (Section IV.1.c).The irradiating RF waves/B1 field are then turned offand the nuclei allowed to relax (Section IV.3) back totheir equilibrium population, which they do by emittingRF waves whose parameters provide information aboutthe sample. These emitted RF waves are collected asFree Induction Decays, or FIDs (Section IV.3) in theTime domain (Section IV.3). The FIDs are mathematicallysimplified and transferred into the Frequency domain, usinga technique called Fourier Transformation (Section IV.4),where they are usually presented in the form of a one-dimensional (1D) or two-dimensional (2D) spectrum—ortransient—containing a number of resonances (Section IV



A

B

Fig. 2. Sample one-dimensional (1D) and two-dimensional(2D) nuclear magnetic resonance (NMR) spectra of yeastmetabolite extracts. (A) A 1D 1H NMR spectrum of yeastmetabolites, showing intensity on the y axis and resonantfrequency (also known as δ or chemical shift, see Section IV.2a)on the x axis. (B) A 2D spectrum of the same sample as in A, withboth axes now representing a different resonant frequency—13Cfor the y axis and 1H for the x axis—and the intensity of thepeak being reflected by the closeness of packing in the contourplot. It should be noted that the y and x axes are often calledby their spectroscopic names of F1 and F2, respectively. Thisspectrum comes from a heteronuclear single quantum coherence(HSQC) experiment, which is used to follow 1H nuclei whichare attached to 13C nuclei. We thank Reza Salek, DuncanMacInnis and Juan Castrillo at the University of Cambridge forallowing 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 willlook in turn at how an NMR experiment extracts informationfrom each of these parameters.

III. SAMPLE PREPARATION

One of the most striking things about NMR is its abilityto acquire information non-invasively and non-destructivelyfrom an exceptionally broad range of samples, whether in vivo

from within living organisms, such as the human body (Houltet al., 1974), ex vivo from isolated organ preparations (Bittl& Ingwall, 1985; Brindle & Radda, 1985) and intact tissuesamples, such as tumours (Griffin et al., 2003a), or in vitro fromhomogenous tissue extracts in solid, liquid or gaseous form.NMR spectroscopy displays this versatility because it involvesnuclear absorption and emission of RF radiation. Since the

A

B

C

Fig. 3. Building free induction decays (FIDs) and one-dimensional (1D) spectra from the four common nuclearmagnetic resonance (NMR) parameters. (A) Nuclei (filled circles)which have been excited to a high energy spin state will relaxback over time (x axis) to a lower energy, ground, state throughthe emission of photons of electromagnetic (EM) radiation(wavy lines). (B) When these EM photons are summed overa population of relaxing atoms, the decay in the intensityof emitted EM radiation over time will produce a FID.FIDs may be described using four parameters: (1) intensity,(2) frequency, (3) half-life, or t1/2, and (4) phase. NMR is one ofthe few spectroscopic techniques which allows information to beextracted 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 convertedinto a plot of intensity versus frequency in the frequencydomain.

nucleus makes up only a very small part of any atom, RFradiation directed into tissue will have only a small chance ofhitting a nucleus and so RF waves will tend to penetrate tensof centimetres into tissue before they are absorbed, allowingboth imaging and spectroscopy inside the human body forexample. Furthermore, because the flipping of a magneticnucleus from one spin state to another involves only a verysmall amount of energy, any tissue irradiated by RF radiationwill suffer very little damage from subsequent heating asnuclei relax back into their original ground state. A techniquesuch as ultraviolet (UV) spectroscopy, by contrast, involvesthe absorption and emission of radiation by the electron



clouds which make up the bulk of an atom’s volume, so UVradiation is absorbed rapidly and cannot penetrate as deeplyinto tissue as RF radiation. Similarly, were microwaves usedthen they would be absorbed and released by the covalentbonds between atoms, leading to increased molecular motionand significant—and damaging—sample heating.

Nonetheless, despite this enormous potential for in vivo

work, the bulk of NMR spectroscopy is carried out onsamples in solution, for reasons which we will consider inSection IV.2c. Some ex vivo samples—such as blood, urineor cerebrospinal fluid—are already in solution and maybe observed almost directly, although care must be taken toensure that these samples are not compromised in other ways.The haemoglobin in blood, for instance, is paramagnetic—itbecomes magnetic in a magnetic field—and so can distortthe applied B0 field enough to broaden resonances in theNMR spectrum to an extent where they overlap and muchmeaningful information is lost, for reasons discussed inSection IV.2. Blood, therefore, is usually analysed as serum orspun down to remove the haemoglobin-containing red bloodcells and leave plasma. When this is done, the osmolarity ofthe serum or plasma must be maintained with salts duringany dilution to preserve structures such as lipoproteins. Otherfluids present their own idiosyncrasies; NMR spectroscopy ofurine can be impaired by the presence of proteins and metalions such as Mg2+ and Ca2+ which bind various metabolitesand broaden their resonances. To overcome this, one canuse chelating agents such as ethylenediaminetetraacetic acid(EDTA), which bind these cations (Nicholson, Buckingham& Sadler, 1983). In addition, the pH of urine samplesmust be fixed by balancing the ratio of sodium phosphate(NaH2PO4) to orthophosphate (Na2HPO4) to prevent thechemical shift of certain resonances varying from sample tosample (Beckwith-Hall et al., 1998).

For in vitro tissue extracts, of course, solubilization is muchless of a problem and sample preparation is able to focuson the efficient extraction of any molecules present. Thisis made easier because, in contrast to methods such asgas chromatography mass spectrometry (GC-MS), NMRsamples do not need chemical derivatization to make themdetectable. A range of extraction methods are availableand have previously been summarized (Le Belle et al.,2002), with a typical extraction protocol reading somethinglike 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 super-

natant removed [. . .], neutralized with 1 M KOH and

freeze-dried. The freeze-dried residue was resuspended

in 650 μl of deuterated water (D2O) containing 2 mM

3-trimethylsilyl-deuterosodium propionate (d4-TSP) as

a reference standard. (Bothwell et al., 2001, p1634).

Perchloric and trichloric acids are a popular choice foraqueous extracts, especially—as in this example—for brain

tissue in which metabolism must be quenched rapidly. How-ever, acid extraction has the unfortunate disadvantage ofoxidizing many metabolites and proteins, so alternativeextraction protocols are also used (Pears, 2007). The mostcommon of these is chloroform/methanol extraction, withother widely used procedures including acetonitrile/waterextraction and methanol/ethanol/water extraction. Thesealternatives have the advantage of partitioning metabolitesamong several distinct fractions—usually a polar, aqueousmetabolite fraction, a non-polar, lipophilic metabolite frac-tion and an intact and unoxidized protein pellet—allowingmore complete extraction of metabolites and better charac-terization of the sample (Fig. 4).

As in the example protocol, above, extracted fractionsare usually diluted into—or freeze-dried and reconstituted

A

B

C

D

Fig. 4. Different nuclear magnetic resonance spectra fromdifferent fractions from a biological tissue. (A) A chloro-form/methanol extraction produces three fractions. (B) Thelower chemical shift region of the 1H NMR spectrum from theaqueous, methanol fraction of extracts from the Hwacheongrice cultivar (Oryza sativa L.). This region of the spectrumcontains aliphatic polar metabolites, including (1) Trigonelline,(2) overlapping sugar resonances, mostly from sucrose, fructoseand glucose, (3) glycerophosphocholine, (4) O-acetyl carno-sine, (5) asparagine, (6) methionine, (7) glutamine, (8) glutamate,(9) acetate, (10) lysine, (11) alanine, (12) lactate and (13) over-lapping resonances from leucine, isoleucine and valine. (C) Anyprotein in the sample is less dense than chloroform and formsa layer between the chloroform and aqueous phases. (D) Thelower chemical shift region of the 1H NMR spectrum from thecorresponding organic, chloroform fraction extracted from thesame sample as B. This fraction contains non-polar metabolites,including resonances from unsaturated fats (15, 16, 19), satu-rated fats (17, 18), common to all fatty acids (20) and unidentifiedmetabolites too close to the baseline (14). We thank Oliver Jonesat the University of Cambridge for allowing us to present thesespectra.



into—solvents in which protons (1H) have been replacedby deuterons (2H). Deuterons produce NMR signals at adifferent frequency to those of protons, so these deuteratedsolvents serve two purposes. First, they lower the solvent’scontribution to the observed 1H spectrum by reducingthe amount of protonated solvent, which is a far fromtrivial effect when we consider that the concentration ofwater-as-solvent is usually around 50 mol l−1, as opposed tomillimolar metabolite concentrations. Solvent effects can alsobe removed by simple solvent suppression pulse sequences,as explained in Section IV.1b.

Second, most modern NMR spectrometers are operatedin a ‘deuterium locked mode’ where the known frequency ofthe deuterium signal is used to calibrate the other frequenciesobserved during the NMR experiment. Deuterium lockingis usually supplemented by adding a chemical shift referencesuch as, for 1H NMR spectra, 3-trimethylsilyl-deuterosodiumpropionate (TSP) or Tetramethylsilane (TMS) to define moreaccurately chemical shifts (Harris et al., 2002). It shouldbe noted that deuterium locking is less common in olderNMR studies, in which external reference standards areused exclusively; this is because older machines were unableto detect the deuterium signal at the same time as theobserved nucleus or, for protein structure determination, theobserved nuclei.

In our example, above, tissue was reconstituted intodeuterated water (D2O), but many other solvents are alsoavailable: deuterated chloroform (CDCl3) is commonly usedfor lipid metabolites, deuterated di-methyl sulfoxide (DMSO)has been used to solubilise components in the humin fractionof soil (Simpson et al., 2007) and a range of deuterated solventsare used during combined liquid chromatography and NMRspectroscopy experiments (Section V.2c), according to thechromatographic separations that are being used (Dunn,Bailey & Johnson, 2005).

We have emphasized sample preparation for one verygood reason: so long as an NMR spectrometer is configuredand operated correctly, multiple runs on the same sampleshow an exceptionally high degree of reproducibility,making high-resolution NMR spectroscopy a more robustanalytical approach than those based on chromatographicand/or mass spectrometry based methods, such as highperformance liquid chromatography (HPLC) or GC-MS.In practical terms, this means that, so long as a standardoperating protocol is followed, researchers can be confidentthat variation between samples reflects biological, and notinstrumental, variation. This is particularly important ifspectra are to be analysed by automated pattern recognitiontools. Indeed, NMR is such a powerful global analyticalapproach that it can often detect unexpected biologicalvariation in a dataset and variations in diet, age, growthconditions, hormonal status and strain background haveall unexpectedly muddied the interpretation of studies inplants, animals and micro-organisms (Bollard et al., 2005;Griffin & Nicholls, 2006; Gulston et al., 2008). The morestandardized the sample preparation can be made, therefore,the better.

IV. DATA ACQUISITION

After preparation, samples are put into the most distinctivefeature of an NMR experiment—the barrel-shapedaluminium magnet—and data acquisition begins. Thefollowing example protocols give an idea of the variousmagnet and spectrometer settings which must now beconsidered:

‘‘All spectra were obtained at 30◦C on a [. . .] spectrom-

eter 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.’’ (Raamsdonket al., 2001, p 50).

As we stated at the end of Section II, the purpose of theseprotocols is to measure four parameters (intensity, frequency,half-life and phase; Fig. 3) of the EM radiation which isreleased when a population of nuclei relax from one spinstate to another. We will look in turn at how an NMRexperiment measures each of these parameters.

(1) Measuring intensity and optimizing thesignal-to-noise ratio

Any spectroscopic technique benefits from a high signal-to-noise ratio and an important limitation with NMR, relativeto other spectroscopies, is its lack of sensitivity. The inherentability of any nucleus to absorb EM radiation is given by itsgyromagnetic ratio: nuclei with higher gyromagnetic ratioswill absorb EM radiation more readily than nuclei with lowergyromagnetic ratios.

Theorists in the late 1920s and early 1930s realized thatthe energy difference between nuclear spin states woulddepend on the strength of the magnetic field in which thenuclei were sitting. Unfortunately, in the Earth’s magneticfield, these energy differences are vanishingly small, so that,even for nuclei with higher gyromagnetic ratios, the smallenergy differences between nuclear spin transitions are of theorder of thermal energy at room temperature. This meansthat ambient heat suffices to move many nuclei into higherspin states, making the population difference between highand low energy spin states very small—typically less than afew parts per million. This, in turn, reduces the number oflow energy nuclei which may be excited in a sample and thusreduces the maximum achievable signal-to-noise.

To overcome this low-sensitivity problem, spectroscopistsuse 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 thesaturation of the sample, (d) they reduce the temperatureof the sample and (e) they reduce the volume in which theNMR signal is observed.

(a) Increasing B0

Since theory predicted that energy differences betweennuclear spin states varied with the external magnetic fieldstrength, and since very low energy EM radiation wasalmost impossible to detect using the electronics availablein the 1930s, Isidor Rabi’s pioneering group at Columbiaplaced nuclei in an extremely strong homogenous B0 field.This increased the energy differences between spin statesso that the nuclei could absorb EM radiation—appliedas a B1 field—in the more easily detectable RF range.These early experiments used molecular beams of nuclei,because these early workers were interested in nuclearphysics, not in probing biological samples. However, EdwardPurcell (Purcell, Torrey & Pound, 1946) and Felix Bloch(Bloch, Hansen & Packard, 1946), working independently atStanford and MIT, soon realized that NMR signals couldalso be obtained from more everyday preparations. Paraffinwax and tubs of water were early favourites; we have alreadyexplained (Section III) that the strong 1H signal generatedby water remains one of the most common complications inbiological NMR spectroscopy and is the reason why samplesare usually reconstituted in deuterated solvents.

Magnetic field strength is usually measured in Tesla, giventhe symbol T. Today’s B0 fields are generated by powerfulelectromagnets which generate fields between 2 and 21 T,several hundred times as strong as the Earth’s own 30–60 mT(Le Mouel, Kossobokov & Courtillot, 2005) field. The actualelectromagnetic coil takes up only a small amount of thedistinctive aluminium barrels, but in order to generate suchhigh B0 fields, superconducting material must be used andthis, in turn, must be cooled to liquid helium temperaturesof around 4 K (−269 ◦C). The bulk of the barrel consists,therefore, of two cooling jackets—a liquid helium one tocool the electromagnet and a liquid nitrogen one to coolthe liquid helium. Furthermore, in modern magnets thesuperconducting B0 coil is surrounded by another coil whichprevents, at least in part, the B0 field from extending outsidethe cylinder, so that spectroscopists can hug a 9.4 T magnetand still be certain that their credit cards will survive.

The first way of improving signal-to-noise would simplybe to increase the strength of the B0 field, to a currentcommercial maximum of 21 T, or 950 MHz, therebyincreasing the population difference between low and highspin states. Unfortunately, this is usually a prohibitivelyexpensive way to increase signal-to-noise ratios, as magnetcosts increase rapidly with field strength.

It should be noted that there is an alternative measureof magnet strength commonly used by spectroscopists.We mentioned in Section I that nuclei which absorb RFradiation of a particular frequency are said to resonateat that frequency, hence the R-for-Resonance in NMR

and hence why absorption peaks are commonly known asresonances. As we have explained, the EM frequency atwhich nuclei absorb, or resonate, depends upon the strengthof the B0 field; in a 9.4 T magnet, for example, 1H nucleiresonate at 400 MHz, in the same range as wireless local areanetwork (LAN) and mobile phones, and 13C nuclei resonateat 100.2 MHz. Magnets are, therefore, often described bythe frequency at which 1H nuclei resonate, so that the samemagnet may equally correctly be referred to as a 9.4 Tmagnet or a 400 MHz magnet, according to taste.

(b) Signal averaging

Second, and very commonly, signal-to-noise ratio can beimproved by averaging together individual spectra, ortransients. However, while signal increases as a functionof the number of transients, n, noise increases as a functionof

√n, which means that the signal-to-noise ratio increases as

n/√

n, or√

n. A two-fold increase in signal-to-noise thereforecomes at a four-fold cost in time, which can soon becomeprohibitive.

(c) Increasing the population difference in a sample

In order to maximize signal-to-noise in, for example, NMRspectroscopy with spin 1/2 nuclei such as 1H and 13C, theexcess nuclei in the ground, −1/2, state should be flipped intothe +1/2 state to give equal populations of nuclei in each spinstate. This process of population equalization is called fullsaturation of the sample and is achieved by applying a 90◦

pulse from the B1 coil; ‘90◦’ reflects the fact that when weredistribute magnetic nuclei in this way, we also move thenet magnetization of the sample from the z axis to the xyplane, i.e. through 90◦.

After saturation, we need to collect all of the emittedRF signal by waiting for the flipped +1/2 nuclei to relaxfully back to the ground −1/2 state, restoring the originalpopulation difference between spin states. Unfortunately, ittakes a long time to collect spectra using both full saturation,using a 90◦ pulse, and full relaxation, using a between-pulse delay—the relaxation delay—long enough to allowfull restoration of the original populations. This is is aparticular problem if many transients are being averagedtogether. For these reasons, spectroscopists usually aim tostrike an optimal balance between minimizing run time andmaximizing signal-to-noise, and they do this by applyingless-than-90◦ RF pulses (often around 60◦) and allowingonly partial relaxation. Most NMR spectra are, therefore,acquired under partial saturation and partial relaxation. Formost purposes, partial saturation is beneficial, although it cancomplicate the quantification of some compounds, especiallythose with long relaxation times, such as the carbonyl 13Cnuclei (R2-13C=O) found in carboxylic acids.

As an aside, saturation is also used to increase signal-to-noise ratio by reducing noise; specifically, noise from solventprotons (Section III). Because the water resonance is so large,it can swamp other resonances and so is removed from thespectrum by irradiating the water resonance continually, so



that the population difference between high and low energystates is zero. This means during any subsequent irradiationby EM radiowaves there are no protons in water moleculesto contribute a net magnetisation for the NMR effect. Thewater resonance has effectively been decoupled from thesample resonances, so this process is known as ‘decoupling’.

Finally, we can see that decoupling is the simplest exampleof a ‘pulse sequence’, since it involves using one RF pulseto suppress water and then a second RF pulse to excite thesample nuclei-of-interest. We will meet more complex pulsesequences later (Section V.2b).

(d) Temperature reduction

The signal-to-noise ratio can be increased by temperaturereduction in two different ways. Either the temperature ofthe sample may be reduced by cooling—although this islimited by the temperature at which the solvent freezes—orthe temperature of the equipment may be reduced usingcryogenic probes (Styles et al., 1984). Sample cooling reducesthermal motion so that more nuclei will sit in lower energyspin states. This increases the population difference betweenground and excited spin states and thus increases signal-to-noise ratio by increasing the maximum signal. Probe cooling,on the other hand, reduces the contribution of electronicand thermal noise and provides a theoretical up-to-four-foldincrease in signal-to-noise ratio. The increased sensitivity ofcryogenic probes has the added benefit of allowing the useof nuclei with lower sensitivity (i.e. lower gyromagnetic ratio)than 1H, which would normally be prohibited due to thetime required to acquire a sufficient signal (Keun et al., 2002).

(e) Volume reduction

Finally, the more homogenous the B0 field, the better thesignal resolution. Since a homogenous field is easier togenerate over a small volume than a large, most magnetshave narrow bores of only a few centimetres, althoughthose used for in vivo work must, of course, have bores largeenough for living organisms to fit inside. These larger boresrange from tens of centimetres for small animals to the widebore medical magnets used for patient studies. Providedthe individual is stationary, the effect of magnetic fields onpatients is minimal, although recently the EU has tried toplace exposure limits for magnetic fields. So little is knownabout magnetobiology, and even whether magnetic fieldscan induce long-term effects, that these limits are, to a largeextent, arbitrary.

Additionally, and for esoteric reasons connected to thegeometry of probe coils, small probe coils are inherentlymore sensitive than larger coils for a given mass of sample,so that microprobes which hold only a few microlitres ofsample can give signal-to-noise ratios which rival cryoprobes(Sakellariou, Le Goff & Jacquinot, 2007). Microprobes arealso useful, of course, when sample volume is limited forbiological reasons, for example in the analysis of the fewmicrolitres of cerebrospinal fluid in the brains of individualmice (Griffin et al., 2002).

Even without the improvements associated withcryoprobes and microprobes, typical sample acquisitiontimes for a 1H NMR experiment where the sample isnot limited are between 6 and 15 min. Furthermore, theapproach is cheap on a per sample basis and thus largedatasets can be rapidly acquired, especially if automation isused, with more than 150 samples in 24 hours being easilyachievable. Hence, the tool has been widely used in the drugsafety assessment area of toxicology during drug screening(Beckwith-Hall et al., 1998).

(2) Measuring energy—chemical shift, or δ

We noted that nuclei of the same element absorb EMradiation of a characteristic frequency in a B0 field (Section I).This is not strictly correct and would make for a pooranalytical technique, as we would only be able to distinguishdifferent elements within a mixture. We should, moreaccurately, have said that nuclei of the same element absorbEM radiation over a characteristic range of frequencies. Thisis demonstrated by the experiment which turned NMR into aversatile analytical tool: the observation that the 1H spectrumof ethanol —CH3CH2OH— consisted of three resonances(Proctor & Yu, 1950). This was unexpected, because it hadbeen assumed that all the 1H nuclei in a sample would absorbRF radiation of the same energy when moving from the −1/2to the +1/2 spin state. Instead, it swiftly became apparent thatthe resonance of a nucleus shifted according to its chemicalenvironment, with ethanol’s three resonances reflecting itsthree 1H-containing chemical groups; -CH3, -CH2- and-OH (Arnold, Dharmatti & Packard, 1951). The discovery ofthis so-called chemical shift, often abbreviated to the Greekletter δ, brought NMR into many chemistry departments asa highly discriminatory analytical probe and this, togetherwith its penetrance and non-invasiveness, meant that it wassoon adopted in the analysis of biological samples (Odeblad,Bhar & Lindstrom, 1956).

31P NMR spectroscopy provides a striking illustrationof how chemical shift may be used in experiments onmuscle function in vivo (Fig. 5A), with 31P chemical shiftdifferences readily allowing the spectroscopist to distinguishresonances from phosphocreatine, inorganic phosphate andATP (Fig. 5B). Notice that, in in vivo spectra from muscle(Fig. 5B), there is no contribution from ADP, which wouldproduce two resonances in 31P NMR spectra. This was animportant early finding of biological NMR, demonstratingthat the concentration of ADP measured in assays in vitro

does not reflect the low intracellular free concentrations ofADP, because ADP binds readily to proteins within thecell and its effective concentration is reduced accordingly(Meyer, Brown & Kushmerick, 1985). Moreover, becausethe inorganic phosphate resonance is actually an average ofmuscle phosphate (H2PO4

−) and orthophosphate (HPO42−),

it is sensitive to changes in pH, producing a change inchemical shift which can be used to measure pH changesduring metabolic insults such as ischaemia (lack of bloodflow), hypoxia (lack of oxygen) or cold (Fig. 5B) (Sartoris,Bock & Portner, 2003).



A B

Fig. 5. The uses of chemical shift: 31P measurement of musclepH in vivo. (A) pH changes play an important role in allowinganimals to adapt to changes in temperature. This has beeninvestigated in two species of eelpout, a small bony fish,using 31P NMR spectroscopy. Sartoris et al. (2003) comparedtwo species : the temperate Zoarces viviparus (shown in A) andthe cold-adapted Pachycara brachycephalum (Pappenheim, 1912).(B) Muscle energetics were assessed in both species to measureintramuscular pH. This is shown for Zoarces viviparus in normalwater (top spectrum) and warm water (bottom spectrum).Intramuscular pH decreases when the fish is heated. Adapted,with permission, from (Sartoris et al., 2003).

As 31P spectroscopy demonstrates, chemical shift is mostuseful when signals from nuclei in different chemicals canbe distinguished. This separation may be optimized in fourways: (a) shimming, (b) sample spinning, (c) using solution-state studies and (d) magic angle spinning.

(a) Shimming

The EM radiation absorbed and emitted by each nucleus is afunction of both the B0 field and the chemical environment,so good signal separation requires both of these to beas homogenous as possible. Because no electromagnet,regardless of cost, can ever generate a truly homogeneousB0 field, and because the actual sample itself disturbs the B0field, the B0 field must be re-homogenized, or shimmed, aftersample introduction; a practice which takes its name fromthe engineering trick of using small wedges of material toadjust the position of a larger object. Shimming used to be atime-consuming manual process, but is invariably automatedon modern machines, allowing the B0 field homogeneity tobe fine-tuned once the sample is sitting in the magnet.

(b) Sample spinning

When combined with the auto-shimming mechanisms onmodern NMR spectrometers, modern electromagnets candeliver acceptably homogenous B0 fields. However, many

older magnets will require the sample to be spun, usuallyat around 20 Hz. This spinning averages out any remainingsmall B0 field inhomogeneties in the horizontal (often referredto as xy) plane, improving the final B0 field homogeneity.

(c) Solution-state studies

The need for field homogeneity also explains the usualuse of samples in solution. In a heterogenous sample,different sample regions will experience different magneticenvironments, most commonly through two phenomenacalled 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 betweentwo spin states, rather than a sharp line. In solution,the free tumbling of solutes means that the chemicalenvironments of those solutes are as similar as possible,which reduces this line-broadening to give distinct, non-overlapping resonances. If shimming, spinning and solutionsare effective, resonances will be narrow and well defined,with a good 1H linewidth—the width of the resonance athalf its height—being less than 1 Hz.

We should point out here that the cell is not a truesolid, but rather a semi-viscous liquid, and its metabolitetumbling rates are fast enough to ensure reasonably narrowresonances for in vivo tissue studies. Furthermore, manytissues are homogeneous enough for the effects of chemicalshift anisotropy and magnetic susceptibility to be ignored toa first approximation. This has allowed clinicians to probeevery organ of the human body by magnetic resonanceimaging and many of these have also been studied by NMRspectroscopy.

(d) Magic angle spinning

Line-broadening is a particular problem with solid tissuesamples, in which compounds cannot ‘tumble’ as freely asthey can in solution. A combination of slow relaxation ratesand magnetic susceptibility and chemical shift anisotropyeffects tends to produce a broad amorphorous resonancein any solid examined by NMR and, at least to a firstapproximation, would suggest that in vivo spectroscopy wouldbe uninformative for following much solid-state biology.However, resolution of resonances in solid samples can beimproved by the process of magic angle spinning (MAS)NMR spectroscopy (Lowe, 1959).

Andrew, Bradbury & Eades (1958) observed that manyof the line-broadening effects in solids depended on theangle at which the sample was placed with respect to the B0field. They went on to demonstrate that rapidly spinning asolid sample at the so-called ‘magic angle’, 54.7◦, resulted inspectra with significantly reduced linewidths. This is causedby a combination of physical effects whose explanation isso complicated that ‘magic’ really is the most descriptiveadjective! Briefly, various physical effects scale according tothe angle at which the sample is placed with respect to theB0 field; at the magic angle the angular term becomes zeroand a narrow resonance is produced. Applying this approach



to a range of biological tissues, in which most metabolitesare in the semi-viscous cell cytosol, linewidths comparableto solution-state NMR spectroscopy have been produced inmany different tumour types and neurological tissues takeneither during surgery or post mortem in humans (Cheng et al.,1997; Griffin et al., 2003b; Millis et al., 1997). This has beenextended further to the spinning of whole organisms, withmice being technically the most convenient (Wind, Hu &Rommereim, 2003).

(3) Measuring relaxation

Nuclei in excited spin states usually relax with distinctivehalf-lives of around 100–1000 ms and, like chemical shift,changes in the exact value of the half-life can give informationabout the environment of the molecules involved. Therelaxation signal is measured as a decay in signal intensityover a few seconds—the free induction decay (Fig. 3)—andwe note in passing that an exactly analogous, althoughless informative, phenomenon is seen for light spectroscopy,in which fluorescence lifetime imaging is used to increasethe information obtainable from visible light excitation offluorescent molecules (Suhling, French & Phillips, 2005).

The NMR signal decays through two mechanisms: T1and T2. The T1 relaxation time (also called the spin-lattice relaxation time) refers to the restoration of theequilibrium population of the spins along the B0 fieldaxis (i.e. the superconducting magnet for most NMRapplications) following the RF pulse. This process takes placelargely through nuclear interactions with the surroundingenvironment and, because it determines how many nucleiare available for excitation, affects the rate at which exciting,B1, pulses can be applied to a sample during an NMRexperiment. By definition, therefore, the T1 relaxationrate determines the extent to which a sample may besaturated during data acquisition: if the T1 relaxation islong, a long relaxation delay is needed between each pulse(Section IV.1c).

The T2 relaxation time (also referred to as spin-spinrelaxation time), on the other hand, refers to the loss ofmagnetisation coherence that creates the EM radiationwhich is emitted during the NMR experiment. This arisesfrom quantum mechanical effects and the homogeneity ofthe magnetic field, and determines how broad the NMRresonances are. For various reasons, this T2 relaxation takesplace in the xy plane (i.e. perpendicular to the magnet) andis always faster than T1 relaxation.

(4) Measuring phase

By the late 1950s, NMR spectroscopy had becomeestablished as a useful analytical technique, with its maindisadvantages, then, as now, lying in its low sensitivityand spatial resolution. This low sensitivity was a particularproblem because spectra were acquired using what wascalled ‘continuous wave’ NMR, in which samples wereexcited using a series of single-wavelength RF waves, eachof slightly longer wavelength than the last, in a manner very

similar to modern visible light spectroscopy. Nevertheless,NMR spectroscopy’s ability to penetrate deeply into samplesand to identify their composition without damaging themmade it particularly well suited to certain in vivo biologicalapplications.

If this were all, NMR would be simply another typeof spectroscopy; a useful, but not essential, addition tomodern biology’s toolkit. There is, however, a particularlyuseful feature of NMR: because RF waves are low energy,and therefore long wavelength, technical considerationsmean that both their intensities and their phases may bedetermined, in contrast to the majority of EM radiationused by biologists, such as the visible and near-visible lightwavelengths, for which only information about intensitiesmay be determined. This means that pulse sequences canbe used to simplify spectra and to add information—visiblelight is only slowly being manipulated in this way with thenew generation of ultrafast pulsed lasers (Jonas, 2003; Krollet al., 2007).

There are two ways in which NMR’s phase informationhas been particularly useful. One of the biggest advances inNMR spectroscopy during the early years of its developmentwas the fast Fourier transform (Ernst & Anderson, 1966)which takes a free induction decay in the time domain andconverts it into the frequency domain to produce a spectrum(Fig. 3). This development significantly speeded up spec-troscopy as it allowed the acquisition of a range of frequenciesin a single pulse, rather than necessitating a laborious scanacross a range of frequencies (as occurs in many UV/visiblespectrophotometers). This leaves more time for the biggestbugbear of most NMR spectroscopists—the inherent lack ofsensitivity of the approach.

The second application of phase has been the use of pulsesequence and multidimensional spectroscopy. This will bedealt with further in Section V.2b.

V. DATA ANALYSIS, OR WHAT CAN THEFREQUENCY DOMAIN TELL US?

The four parameters of the emitted EM radiation collectedfrom an NMR experiment—intensity, frequency, relaxationand phase (Fig. 3)—contain a great deal of information andNMR spectroscopy therefore provides a highly quantitativeand discriminatory analytical tool, capable of distinguishingmetabolites in a mixture, or individual amino acids ina protein. Furthermore, multidimensional outputs can begenerated to show the dependence of one parameter on one,two or all three of the others, and these inter-relationshipscan shed light on a wealth of other information, includingthe environment the molecules are found in and whetherthey are bound to proteins or to other macromolecules.

Broadly speaking, the information from an NMRexperiment can tell us four things about a sample: (1) howmuch of it is present, (2) what it is, (3) where it is and (4) whatit is doing. We will now examine some examples.



(1) How much is there? Metabolic foot- andfinger-printing

The simplest, and most common, type of NMR spectroscopyproduces a spectrum of intensity on the y axis againstfrequency on the x axis—a so-called 1D plot, or frequencydomain (Fig. 3). This is analogous to the intensity versuswavelength plots generated by other forms of spectroscopy.Resonances in 1D plots are mathematically multiplied in aprocess called line-broadening, which simplifies subsequentquantification by smoothing the raw data to reduce thecontribution noise makes to the resultant NMR spectrum.Because the absolute frequency at which nuclei resonate isprimarily a function of the applied B0 field, resonances arenormalized by plotting the x axis as a shift from a referencestandard, so that published chemical shifts are independent ofthe magnet strength and so that spectra acquired in differentB0 fields may be compared. Indeed, the use of such a relativefrequency scale is even needed to compare results betweenmagnets of the same nominal B0 field strength. In order toensure that magnets of a given strength do not interfere withone another if placed in the same facility they will have asmall offset in terms of the magnet field strength. These B0differences between apparently equivalent magnets are oftengreater than proton chemical shifts.

As mentioned in Section IV, chemical shift differencesare usually small, so that if the 1H nuclei in the externalshift reference, TSP (Section III), resonate at 400 MHz ina 9.4 T magnet, the C1 1H nuclei in glucose will resonateat 400.0021 MHz. Chemical shift is, therefore, given inparts per million (ppm) so that, in this particular example,glucose would have a set of C1 1H resonances centred at1,000,000 ∗ (400.0021 − 400)/400 = 5.25 ppm. Any givenexperiment 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 ofthe molecules which have contributed to the spectrum.As we noted in Section IV.1c, one of the ways in whichsignal-to-noise ratios are increased is by performing NMRspectroscopy in a mode that is termed partially saturated,where nuclei are excited at a quicker rate than would allowthe NMR signal to decay completely to zero. Fortunately,saturation factors can be calculated readily, so a simplelinear relationship exists between the area under a partiallysaturated peak and the concentration of the nuclei thatproduce the signal. This gives us a characteristic metabolicphenotype from every sample—akin to a barcode, or asnapshot—which can be statistically separated from anotherset of samples using multivariate analysis such as principalcomponents analysis, even if the individual resonancescannot be identified (Bundy et al., 2007; Raamsdonket al., 2001).

This snapshot approach has proved itself to be particularlyuseful in functional genomic studies, in which genes arealtered and the phenotype of the resulting mutants isexamined to provide clues about gene function. Phenotypeshave 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 apparentlysilent mutations, as other regulatory processes compensateto 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 sensitivephenotype. If the metabolic snapshot comes from cell,tissue or organism preparations, this approach is known asmetabolic fingerprinting; if the snapshot looks at cell mediaor excretory products, it is known as metabolic footprinting(Allen et al., 2003).

Both finger- and footprinting work best with sensitiveand abundant nuclei, such as 1H; one of the most famousexamples of this approach is the highly cited functionalanalysis by co-responses in yeast (FANCY) manuscriptby Raamsdonk et al., 2001. This work showed that anumber of Saccharomyces cerevisae (yeast) strains with similargrowth rates had markedly different 1H NMR spectrawhich could be used to distinguish glycolytic mutantsfrom oxidative phosphorylation mutants. FANCY hasrecently been extended to identify metabolic modulesin S. cerevisiae (Bundy et al., 2007). Briefly, extracellularmetabolite profiles, metabolic footprints, were quantifiedby 1H NMR spectroscopy and analyzed using principalcomponents analysis (PCA) (Fig. 6). This is an unsupervisedstatistical analysis technique that fits a large multivariatedataset onto a multidimensional set of orthogonal axes. Theseaxes, which are called principal components, are chosen sothat each axis explains as much of the variance remaining inthe dataset as possible. The overall aim of PCA is to reducethe number of parameters needed to describe the differencesamong datasets and, properly applied, PCA is a powerfulway to classify mutants and their associated genes (Fig. 6).

As well as the convenience of not having to identifymetabolites, snapshot approaches can also be used in highthroughput screens where metabolite identification can bepostponed until an effect has been observed. Using suchan approach, the consortium for metabonomic toxicology(COMET) investigated around 150 model liver and kidneytoxins during a three-year study using NMR-based analysisof urinary metabolites (Lindon et al., 2005; 2003). It ishoped that such an approach will allow the generationof expert systems where liver and kidney toxicity can bepredicted for model drug compounds, with the databasesbeing easily transferable among laboratories. In addition theapproach has also been used to screen human populationsto understand disease processes (Brindle, 2002; Kirschenlohret al., 2006) and monitor the effects of diet (Lenz et al., 2004).

This metabolomic approach to analyzing gene functionhas three advantages over gene- or microarray-basedapproaches. First, it measures functional entities, whichreduces biological noise. Second, there are a smaller numberof metabolites than there are genes. Which simplifies analysis:at the last count, around 1500 metabolites were found inthe yeast S. cerevisiae as opposed to 6000 genes (Goffeau et al.,1996; Herrgard et al., 2008). Indeed, the components of



0.6PUT3

PUT1

URA3

PPR1

URA2URA10

URA5

URA8

URA7

PUT2HO

1.0Glucose

0.5

0.0

-0.5

-1.0

0.4

0.2

0.0

PC

2

PC

1 lo

adin

gs

PC1

-0.2

-0.4

-0.6

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

Variable order

A B

Fig. 6. Metabolic footprinting of yeast mutants. (A) principal component 1 (PC1) (x axis) versus principal component 2 (PC2) (y axis)scores plotted for 1H nuclear magnetic resonance (NMR) footprints from eleven Saccharomyces cerevisiae yeast strains: three prolineutilization 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 URA7and URA8 sit well away from the others, on the right of the plot. (B) The difference between URA7 and control spectra is plottedas a loadings plot, with the variable order (the pattern recognition software’s term for chemical shift) on the x axis. Loadings plotshighlight which metabolites are most discriminatory in the PCA plot. In this example, the regions containing glucose are all shownto have increased in the loading for PC1, demonstrating that URA7 has increased glucose concentrations relative to the controlstrain. Taken together, these results suggest that the URA7 and URA8 gene products are subject to different regulatory controlsthan the other genes. Adapted, with permission, from Bundy et al., (2007).

central metabolism which dominate the metabolome onlynumber a hundred or so metabolites. Third, it is context-dependent because it can measure how the plasticity of amutant phenotype in response to environmental and externalstimuli.

When considering large-scale analyses of biological mix-tures, the main alternative to NMR spectroscopy hastraditionally been mass spectrometry. Mass spectrometryis undoubtedly the more sensitive technique and in tissueextracts in which 30–60 metabolites might be detected byNMR, a relatively cheap GC-MS instrument will often findmore than 100 (Atherton et al., 2006), with the upper limitto metabolite numbers thought to depend on the constraintsof cellular osmolarity (Krishnan, Kruger & Ratcliffe, 2005).However, despite recent improvements in mass spectrom-etry sensitivity and robustness GC-MS still identifies lessthan 20% of the metabolites present, and methods suchas liquid-chromatography-based MS and Fourier transformMS, as well as niche techniques such as Coulombic arrays,fare little better. The fact that these techniques can still mea-sure only a small proportion of the total metabolome (seeEllis et al., 2007) for a broad review of metabolomictechniques suggests that there are problems associated withmetabolite extraction and resolution, rather than techniquesensitivity.

In any case, because NMR analyses are reproducible, candetect certain critical metabolites which define the regulatorymetabolic 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, ratherthan in competition, to maximize the coverage of themetabolome.

(2) What is it? Metabolic profiling and protein NMRspectroscopy

Metabolic footprinting assigns a quantity to each peak, but1D spectra of the frequency domain also carry qualitativeinformation about a sample. We noted in Section IV.2 thatthe spectrum of ethanol gave three distinct resonances and,in general, each chemical will have its own characteristicpattern of peaks. There are a number of ways in whichwe can identify these resonances, drawn from analyticalchemistry. These are: (a) chemical shift, (b) multidimensionalNMR, and (c) hyphenated NMR.

(a) Chemical shift and spin-spin coupling

As we noted in Section IV.2, the energy difference betweenspin states is dependent upon nuclear environment, soexcited nuclei from one element, but in different nuclearenvironments, will emit slightly different RF wavelengthsof EM radiation. In NMR, the energy of the emittedEM radiation is expressed as the chemical shift, δ, andthe exact value of δ gives a reasonable indication of themolecule’s identity.

This identification may be narrowed down still furtherif we consider each resonance’s splitting pattern. Manyresonances at a given chemical shift will be split into two,



three, or more sub-peaks, called, respectively, doublets,triplets and multiplets. These splittings arise through aphenomenon called spin-spin coupling; each nucleus behavesas a tiny magnet within the molecule, so that nucleus A willaffect the energy of nucleus B, and vice versa, so long asthey are positioned close to one another. Because eachmolecule has a characteristic number and pattern of thesesub-peaks, a simple comparison of unidentified peaks withstandard reference spectra will often allow identificationof the metabolites in a sample. Accordingly, a numberof NMR spectral databases have been set up to aid thismetabolite assignation; these databases include the HumanMetabolome Database (Wishart et al., 2007) and the MadisonMetabolomics Consortium Database (Cui et al., 2008), as wellas a number of proprietary software tools.

(b) Multidimensional NMR

The previous section showed that, in theory, unknowncompounds in a spectrum can be identified by theirchemical shift and by the number and pattern of theirsub-peaks in the frequency domain. Unfortunately, becausethe differences in chemical shift are relatively small for 1H-containing molecules, biological 1H NMR spectra are oftencluttered with many overlapping resonances that make peakidentification difficult: we say that 1H peaks are ‘poorlydispersed’. This dispersion problem occurs in most forms ofspectroscopy, but highlights one of the strengths of NMR;because it is possible to shape RF waves and measure theirphase, and because it is also possible to detect the nuclei ofdifferent elements simultaneously in a single experiment, we

can use RF pulse sequences to add information to a spectrumwhich might help with assignation.

For example, one of the most efficient methods fordetermining which compounds are in a mixture is to useheteronuclear pulse sequences which detect which 1H nucleiare connected to which 13C nuclei. A variety of such pulsesequences exist, which have unfortunately opaque nameslike heteronuclear single quantum coherence (HSQC), het-eronuclear multiple quantum coherence (HMQC) andheteronuclear multiple bond coherence (HMBC) (Braun,Kalinowski & Berger, 1998). These 1H-13C heteronuclearpulse sequences are especially useful because they combinethe relatively high sensitivity of 1H NMR spectroscopy withthe increased dispersion of 13C NMR spectroscopy, the lat-ter having a chemical shift range of approximately 250 ppmcompared with approximately 15 ppm in 1H NMR spec-troscopy. Thus by detecting a combination of 1H and 13Cresonances in a metabolite, we can rapidly narrow downthe identity of the compounds that could be present in thatmixture.

For instance, in order to understand tissue energetics,it is important to know the relative levels of high-energy phosphates. The sperm of many animals containhigh levels of ATP, but mammalian sperm do not,containing instead phosphodiesters and phosphomonoesters.Because phosphomonoester resonances are poorly disperse,multidimensional heteronuclear NMR spectroscopy hasbeen used to identify the exact species of phosphomonoesterpresent and to further our understanding of mammalianfertilization and reproduction (Fig. 7).

A B

Fig. 7. Pulse sequences in multidimensional nuclear magnetic resonance (NMR) spectroscopy. (A) Two-dimensional (2D) 1H, 31Pheteronuclear multiple bond correlation (HMBC) NMR spectroscopy of lyophilized boar sperm extracts. HMBC is a pulse sequencewhich detects 1H nuclei separated by a given number of bonds from 31P nuclei and, in this case, shows a chemical shift correlationbetween the 31P phosphomonoester peak (PME) and a given 1H peak at around 4 ppm. The inset shows an expansion for the PMEcross-peak ; glycerol 3-phosphorylcholine (GPC) and inorganic phosphate (Pi) peaks are also labelled on the 31P spectrum. (B) 2D1H, 13C gradient-selected heteronuclear single quantum coherence (HSQC) of boar sperm extracts. HSQC is a pulse sequencewhich observes 1H nuclei attached to 13C nuclei and this spectrum shows that the 1H peak identified in A is linked to an adeninebase, which identifies the major phosphomonoester component as AMP. Adapted, with permission, from Kalic et al., (1997).



The field where multi-dimensional NMR spectroscopy hasmost impressively been used is its use in the determinationof protein structures as first suggested by among others KurtWuthrich (Wuthrich, 1990). Proteins may have thousandsof NMR resonances associated with them, and thus simpleone-dimensional 1H NMR spectra are highly congested. Tocircumvent this dispersion problem, spectra from proteins areusually collected across multiple dimensions often associatedwith different nuclei. In particular, because the backboneof peptides consists of amide linkages, the use of 1H, 13Cand 15N multi-dimensional NMR spectra is common inprotein NMR spectroscopy. These experiments can consistof two-dimensional NMR spectra such as 1H-13C HSQC and1H-15N HSQC experiments which take hours to 2–3 daysto acquire depending on the protein, whether it has beenartificially labelled with amino acids containing 13C and 15Nand the field strength of the magnet used. Alternatively,more time-consuming three-dimensional experiments canbe performed that simultaneously measure the interactionsbetween 13C, 15N and 1H or experiments. Furthermore,there are certain NMR approaches that do not rely onmagnetisation being carried through bonds as occurs in theHSQC experiment but move through space. The nuclearOverhauser effect spectroscopy (NOESY) pulse sequence isused to measure how close atoms are in proteins and thususeful in placing distance constraints on the size and shapeof a protein. The field of protein spectroscopy is a large andcomplex one and we direct the interested reader to specialistreviews in this area, notably Wuthrich (1990), Reid et al.,(1997) and Shin, Lee & Lee (2008).

(c) Hyphenated NMR

Multidimensional NMR adds another dimension (ordimensions) to a spectrum and effectively adds moreinformation to aid peak assignation. This extra dimensiondoes not have to be an NMR one and it is often desirableto combine NMR with other techniques. For example,we can include online chromatographic separation, whichfractionates the sample before running a sequence ofNMR spectra. This is analogous to GC-MS and liquid-chromatography mass spectrometry (LC-MS). In addition toseparating metabolites according to their chromatographicproperties, liquid chromatography also concentratesmetabolites into chromatographic peaks. By increasing thelocal concentrations of metabolites, the sensitivity of theNMR experiment can be increased (Bailey et al., 2002).

(3) Where is it? Metabolite environment

Differences in chemical shift reflect differences in theenvironment of a nucleus. So far, we have assumed thatdifferent environments mean similar nuclei in differentmolecules. However, they may also reflect the samemolecules in different environments. A number of propertiesof the spectral peak may be affected by environment. Mostsimply, the chemical shift may change, as is the case withthe inorganic phosphate peak in 31P spectroscopy, whose

chemical shift is pH dependent (Section IV.2 and Fig. 5).However, the relaxation times T1 and T2 (Section IV.3) andthe diffusion properties (which can be measured by NMR)can also be used to provide information about molecularenvironments.

For relaxation times, both T1 and T2 are influencedby how fast individual metabolites tumble in the B0field. Metabolites in a restricted environment, and whichcannot tumble freely, produce broad resonances whichare most easily distinguished by direct measurement ofT1 and T2 relaxation times. This approach has beenused to demonstrate the restrictive environment of themitochondrion for both proteins (Haggie & Brindle, 1999)and metabolites (Bollard et al., 2003). In addition, so-calledrelaxation editing has been used to distinguish differentmetabolites in complex biofluids such as plasma using pulsesequences (Liu, Nicholson & Lindon, 1996).

An alternative to relaxation-based approaches is todistinguish metabolites by their diffusion properties. Thisapproach uses a magnetic gradient in which the B0 fieldvaries, usually linearly, across the sample. A second B0gradient is then applied that is equal in magnitude butopposite in sign. For metabolites that do not move rapidlythese two magnetic gradients will cancel out exactly.However, the nuclei of rapidly moving metabolites willreceive a net magnetisation which will vary according tothe position of the nuclei. Averaging over the whole sample,this results in a broadening of the resonance. This approachhas also been used to distinguish low molecular weightmetabolites from lipoproteins and bound metabolites inblood plasma and biological tissues such as intact tumoursusing 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 changesin peak intensity over time to give an idea of the timecourseof metabolite reactions in a way which is not too different tothe use of radiolabels or similar metabolite tags. However,NMR is once again set above other forms of spectroscopy byour ability to measure the phase of EM radiation and thusto use pulse sequences. In particular, using an experimentreferred to as magnetization transfer, NMR spectroscopymay, effectively, introduce a metabolite label at any pointduring the course of an experiment (Bittl & Ingwall, 1985;Degani et al., 1985). For example, magnetization transfer hasbeen used to examine the transfer of phosphate betweenphosphocreatine and ADP (Brindle & Radda, 1985). Here,the phosphocreatine phosphorus nucleus is irradiated byRF waves in such a manner that its population of spins iscancelled out (and hence the resonance would be invisible inthe NMR effect). When this phosphate group is transferredfrom phosphocreatine to ADP to produce ATP, it carriesits magnetisation with it. This shows up as a reduction inthe signal of the ATP γ phosphate. The rate of reductionin the intensity of the ATP γ phosphate resonance can thenbe used to estimate the activity of the creatine kinase which



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 bindingdetails of large protein complexes. Binding of substrates willinduce changes in the structure of the enzyme and this inturn will alter the chemical shift of the resonances observed inthat protein, especially of those involved in the binding site.For instance, Velyvis et al., (2007) studied ligand binding tothe 306 kDa aspartate transcarbamoylase (ATCase) enzymeusing highly deuterated, 1H,13C-methyl labelled ATCasetogether with methyl-transverse relaxation optimized NMRspectroscopy. Previous research had demonstrated thatthe cooperative binding of active site ligands followsthe Monod-Wyman-Changeux (MWC) model of allostery,which proposes the existence of two conformations of anenzyme in equilibrium, T and R. The T form has a loweraffinity for substrates, and as more sites in the enzyme bindsubstrate, the equilibrium shifts toward the R state. Theauthors established that for ATCase, which catalyzes thefirst step in pyrimidine biosynthesis, only the T state of theenzyme can be observed in NMR spectra of unligandedATCase. However, the binding of nucleotides such as ATPor CTP shifts the T-R equilibrium so that correlations fromthe R state become visible, consistent with the MWC model,with these resonances shown in black in Fig. 8. This studyemphasizes the utility of modern solution NMR spectroscopyin understanding protein function, even for systems with highmolecular masses (Velyvis et al., 2007).

Finally, in just the same way that 14C-labelled com-pounds have been used to determine substrate metabolism inperfused tissues and the whole organism, 13C-labelled com-pounds can be used and detected by 13C NMR spectroscopy.Although 13C NMR spectroscopy is of inherently low sen-sitivity when compared to 1H NMR spectroscopy—13C’slow gyromagnetic ratio making it an order of magnitude lessdetectable than 1H—it can be performed in vivo or in situ inreal time allowing the measurement of the rates of processessuch 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 compartmentationwithin 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 beingaddressed for some applications involving 13C NMRspectroscopy, thanks to a resurgence of interest in a techniquecalled hyperpolarization (Golman et al., 2003; Schroederet al., 2008), in which huge population differences can bebuilt up between ground and excited spin states in certainmolecules. In hyperpolarization, the sample is mixed witha polarization transfer agent before being frozen solid. Thepolarization transfer agent is typically something with anunpaired electron which can be excited by microwaveradiation. This, incidentally, is electron spin resonance,

Fig. 8. Protein spectroscopy. Two-dimensional (2D) 1H-13Cmethyl-transverse relaxation optimized spectroscopy (TROSY)heteronuclear multiple quantum correlation (HMQC) spectrumof U-[2H] Ile-[δ113CH3]-labelled aspartate transcarbamoylase(ATCase) recorded at 800 MHz, showing 27 resonances whichcorrespond to the 27 Ile residues in ATCase. TROSY is a pulsesequence that specializes in the analysis of large proteins. Fornormal one-dimensional (1D) and 2D spectroscopy, resonancelinewidths increase with molecular mass, producing short T2relaxation times, broad resonances and correspondingly poorresolution. TROSY avoids this line-broadening and producesnarrow peaks for large proteins, allowing the discrimination ofresonances not possible using other pulse sequences. By wayof demonstration, the arrows point to the cross-peaks throughwhich the four 1D traces have been drawn. These four tracesshow the high signal-to-noise ratio and illustrate the sensitivityof the spectrum, which comes from solution studies and the veryhigh B0 field strength (800 MHz). Peaks from the r and c chainsof ATCase are colored red and black, respectively, with thechange in structure induced by ligand binding. Adapted, withpermission from (Velyvis et al., 2007).

which is analogous to NMR except that it occurs at a higherEM frequency, since electrons have larger energy differencesbetween their two spin states. In the solid, frozen, statemagnetization is transferred from the polarisation transferagent onto the sample. The sample is then rapidly thawedbefore being used for conventional NMR spectroscopy.Hyperpolarization can build up huge population differences,but with the disadvantage that differences begin to disappearthrough T1 relaxation as soon as the sample is thawed(Golman et al., 2003). As a result most applications have sofar involved carbonyl 13C resonances, as these tend to haverelatively long T1 times (tens of seconds) and thus allowthe magnetization to be maintained during the experiment.Despite this limitation, this approach has been used tofollow metabolism in tumours and heart tissue with exquisitetemporal resolution (Fig. 9).

VI. CONCLUSIONS

(1) NMR spectroscopy is a powerful and flexible techniquewhich allows biologists to study the molecular and



A

B

Fig. 9. Hyperpolarization. (A) An example time course ofspectra acquired from a rat heart in vivo over 60 s ofdata acquisition. The arrival of [1-13C]pyruvate can beseen, followed by its subsequent decay back to thermalequilibrium levels. The appearance of the metabolic products,[1-13C]lactate, [1-13C]alanine and bicarbonate (H13CO3

−)follows shortly afterwards through [1-13C]pyruvate metabolism.(B) An example of the temporal variation in the fitted peak areasof each metabolite over the 60 s time course. The pyruvate areahas been reduced by a factor of 10 to improve visualization.This figure was supplied to the authors by Damian Tyler at theUniversity of Oxford.

cellular environments of magnetic nuclei. Commonlystudied magnetic nuclei include 1H, 13C and 31P.

(2) In NMR spectroscopy experiments, magnetic nucleiare placed in a strong B0 electromagnetic field andexcited from lower energy spin states to higher energyspin states by the absorption of EM radiation generatedby a second, B1, electromagnetic field. These excitednuclei then relax by emitting EM radiation of slightlyless energy; both exciting and emitted radiation are inthe RF range.

(3) Because NMR spectroscopy uses low-energy RFradiation which is absorbed by nuclei, it can penetratedeeply into tissue and is non-invasive.

(4) Unfortunately, the low energy of RF radiation alsomakes NMR spectroscopy a relatively insensitivetechnique. There are a number of practicalways around this low-sensitivity problem, includinghyperpolarization.

(5) The emitted RF radiation has four parameters whichmay be analyzed for information about the sample:intensity, frequency, half-life and phase.

(6) The intensities of peaks in an NMR spectrum reflectmolecular concentrations, so NMR spectroscopyis often used as a standard, automated, high-throughput analytical technique, especially suitablefor metabolomics.

(7) The frequencies, or chemical shifts, of peaks inan NMR spectrum can be used to identify whichcompounds are present in a mixture.

(8) The half-lives, or relaxation times, of peaks in anNMR spectrum can give information about molecularenvironments.

(9) NMR spectroscopy has more analytical power—and ismore complicated—than other forms of spectroscopybecause we are able to measure the phase of RFradiation. This allows us to study the connectivitiesbetween magnetic nuclei, giving a tool with which tostudy protein structure and dynamics.

(10) We have focussed mainly on small-moleculespectroscopic applications, despite huge literatureson magnetic resonance imaging and protein NMRspectroscopy. However, we hope that interestedreaders will now be armed with the key conceptsof NMR spectroscopy and will be in a position tobenefit from more specialised reviews and primarypublications.

VII. ACKNOWLEDGEMENTS

J.H.B. is funded by a Leverhulme Early Career Fellowship.Additionally, we are grateful to the BBSRC, MRC, NERCand Royal Society for financial support. We would like tothank Reza Salek, Duncan MacInnis and Juan Castrillo atthe University of Cambridge for the yeast HSQC spectrumin Fig. 2, Oliver Jones at the University of Cambridge forthe rice spectra in Fig. 4, Franz-Josef Sartoris at the Alfred-Wegener-Institut for the eelpout spectra in Fig. 5, KevinBrindle at the University of Cambridge for the yeast spectra inFig. 6, Gunter Kamp at Johannes Gutenberg University forthe sperm spectra in Fig. 7, Lewis Kay and Algirdas Velyvisat the University of Toronto for the ATCase spectrum inFig. 8 and Damian Tyler at the University of Oxford for thespectra in Fig. 9.

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