Essays in Biochemistry (2016) 60 419–428

DOI: 10.1042/EBC20160044

Solution state nuclear magnetic resonance

spectroscopy for biological metabolism and
pathway intermediate analysis
1 1, 2
Gareth L. Nealon and Mark J. Howard
1 Centrefor Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley, Perth, Australia, 6009; 2 Department of Chemistry and Biochemistry,
The University of Western Australia, Crawley, Perth, Australia, 6009
Correspondence: Mark J. Howard (mark.howard@uwa.edu.au)

Using nuclear magnetic resonance (NMR) spectroscopy in the study of metabolism has been
immensely popular in medical- and health-related research but has yet to be widely applied
to more fundamental biological problems. This review provides some NMR background rel-
evant to metabolism, describes why 1 H NMR spectra are complex as well as introducing
relevant terminology and definitions. The applications and practical considerations of NMR
metabolic profiling and 13 C NMR-based flux analyses are discussed together with the el-
egant ‘enzyme trap’ approach for identifying novel metabolic pathway intermediates. The
importance of sample preparation and data analysis are also described and explained with
reference to data precision and multivariate analysis to introduce researchers unfamiliar with
NMR and metabolism to consider this technique for their research interests. Finally, a brief
glance into the future suggests NMR-based metabolism has room to expand in the 21st
century through new isotope labels, and NMR technologies and methodologies.

Received: 17 June 2016
Revised: 7 October 2016
Accepted: 10 October 2016
Version of Record published:
15 December 2016
Introduction
Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry have become highly influential
technologies over the last 20 years in the study of metabolomics in health and disease. There are many
excellent examples and reviews on NMR-based metabolic studies in clinical settings and readers are en-
couraged to indulge their interests in this area through these associated references [1–5]. However, the
perceived complexities associated with experimental procedures, sample preparation and data analysis
have provided a barrier to many biochemical and biological laboratories who might otherwise be inter-
ested in using this incredibly useful and accessible resource more widely across non-clinical research.
The popularity of studying metabolism has expanded rapidly over recent years to become one of sev-
eral keystone ‘-omic’ technologies. It should be considered complimentary to genomics, transcriptomics,
proteomics and lipidomics where they underpin systems biology and provide a multifaceted interdis-
ciplinary portfolio. As a result, it is imperative to embrace these technologies to maximise impact and
remain at the forefront of bioscience research, but it is inefficient for a group leader to be a master of all
methods. However, regardless of speciality, researchers need a comfortable level of understanding of these
tools to amalgamate techniques efficiently. This article aims to introduce metabolic NMR and encourage
bioscientists to consider discussing potential projects with a collaborative NMR spectroscopist.
Nuclear magnetic resonance spectroscopy utilises samples that contain nuclei with non-zero magnetic
moments that absorb electromagnetic radiation (r.f) at a precise frequency when placed in an external
magnetic field. This is the point where most non-NMR interested scientists switch off but this definition
underlines the fundamental utility of the technique with respect to metabolomics. First, local electronic
environments in a molecule induce small, but measurable, changes in the precise frequency of r.f absorp-
tion; this provides the familiar NMR spectrum of chemical shifts by which different chemical species can
be distinguished. Second, the precise frequency means different nuclei can be ‘tuned’ for observation by an


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NMR spectrometer; this provides the commonly encountered 1 H or proton spectrum as well as other nuclei such as
13
C, 15 N and 31 P. Third, the non-zero magnetic moment makes NMR isotope specific; hence the terminology of 1 H,
13
C, 15 N and 31 P. NMR is selective regarding isotopes and is only possible when the magnetic moment (sometimes re-
ferred to as spin) of these isotopes is non-zero. Furthermore, only spin 1/2 nuclei provide high-resolution NMR spectra
that allow the deciphering of chemical shifts and quantitative detail. As always, caveats exist with these definitions but
it should come as no surprise that 1 H, 13 C, 15 N and 31 P isotopes all have spins of 1/2. Spin also explains the complexity
of isotopic enrichment; 1 H (protium) works well as this primary NMR observable isotope of hydrogen because it is
99.99% abundant, so any molecules containing hydrogen should provide plenty of 1 H NMR signal. This is also the
case for 31 P NMR although phosphorous is more eclectic in its role across metabolism with only a small proportion
of metabolites being phosphorylated. However, the 13 C isotope of carbon is 1.07% abundant with the bulk of carbon
being present as the 12 C isotope that has zero spin and is NMR inactive. Nitrogen is even more complex because the
common 14 N isotope has a spin of 1, but is an excellent example whereby a spin > 1/2 is ‘bad for detailed NMR’, forcing
us to resort to the less common spin 1/2 15 N isotope that is only 0.36% abundant. Consequently, many biological-based
NMR experiments use isotopically enriched 15 N; i.e. this isotope is boosted to excess of 90% in a sample. This comes
at a cost and a logistical design headache regarding how the 15 N isotope is introduced into the experiment or sample.
However, the 13 C isotope is more forgiving and can still be detected by NMR at natural abundance levels, although
isotopic enrichment of 13 C is a possible way of improving metabolism NMR data.
The logical starting point is 1 H NMR because metabolites and pathway intermediates are hydrogen rich; for exam-
ple, sucrose has the molecular formula of C12 H22 O11 and even hydrogen ‘starved’ pyruvate is C3 H3 O3 . As outlined
above, local structural differences in each metabolite alter the local electron environment to create 1 H NMR peaks
that reflect each distinct hydrogen environment. This results in a fingerprint of chemical shifts for each metabolite
that can be unique in many cases, as shown in Figure 1A for the amino acid alanine [CH3 CH(NH3 ) + COO − ]. The
alanine spectrum is acquired such that labile NH3 + protons are not observed and the two peaks are from the CH (left)
and CH3 (right) environments. This sounds promising until one considers the large numbers of unique metabolites
in a cell, media preparation or biological system; adding these collective ‘fingerprints’ explains the crowded nature
of a 1 H NMR profile spectrum, as shown in Figure 1B for a cell extract of Saccharomyces cerevisiae. This article
focuses on the use of high-resolution solution state NMR to investigate metabolites, like those found in the cell ex-
tract in Figure 1, but solid samples such as cells and tissues can be interrogated using high-resolution magic angle
spinning (HR-MAS) techniques for clinical and other specific biological applications [3,6–8]. NMR-based medical
metabolomics involves the study of complex biological fluids such as urine, plasma and blood, and forms the basis of
improving health and diagnosis through metabolic phenotyping with National and International Phenome Centres
being established across the globe.
As most metabolites have more than one ‘H’ environment, each metabolite spectrum is complex in its own right.
Once multiplied by the number and variety of metabolites, it is now much easier to understand why a cell extract
spectrum is extremely busy. The subject of crowded spectra leads to the choice of spectrometer. NMR spectrometers
are generally referred to by their r.f. carrier frequency for proton (1 H) at their particular magnetic field; 600, 700,
800 etc. corresponds to the carrier proton frequency in megahertz (MHz) at magnetic field strengths of 14.1, 16.5
and 18.8 tesla respectively. NMR spectrometer resolution goes hand-in-hand with frequency; therefore, using higher
field spectrometers increases peak separation and allows more metabolite peaks to be unambiguously identified. As a
guide, a 600 MHz system is a good workhorse but if you have access to 700, 800 or 950 MHz then use them. However,
do not be deterred by the complexity of NMR spectroscopy or the data it provides because the spectrum in Figure
1B is a metabolic profile in its own right and deviations from this profile can be scrutinised in two ways; analytically,
for specific changes in individual metabolites or holistically, to observe trends and changes as a result of a biological
process or event.

Metabolic profiling
As with all research, the choice of experimentation and analysis is project specific and should always begin with an
understanding of all questions to be answered. For example, if your interests revolve around a cellular or biological
process that may generate a metabolic response, start with a metabolic profiling study using either cell contents or
media extract, depending on whether the response is intracellular or extracellular.
Profiling is extremely powerful because it doesn’t require any prior knowledge of any potential metabolic changes
during the initial study, although if suspicions exist, specific metabolites can be targeted for analysis. As it is important
to assess whether significant metabolic changes occur between two or more conditions or processes, and that these
changes may relate to controllable genetic, proteomic or lipidomic differences, this can provide a systems biology link

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H2O CH3

CH2
(A) TSP

TSP

H2O
(B)

8 6 4 2 0
1
H chemical shift (ppm)

Figure 1. 1 H NMR spectra of alanine (A) in water (using a pulse program to reduce the residual signal from H2 O) and S.
cerevisiae cell extract (B) in D2 O (heavy water) acquired at 25◦ C on a 14.1 tesla (600 MHz 1 H) Bruker AV3 NMR spectrometer
with a QCI-F cryoprobe.
Alanine CH2 and CH3 proton peaks are labelled together with residual water resonance (H2 O) and internal reference standard; 20 μM
3-(trimethylsilyl)propionate (TSP).

to your study. In the first instance, consider two conditions of interest and observe the NMR metabolic profiles for any
changes. Examples include observing profile differences between wild-type and mutant strains, growth conditions or
even media types. This is illustrated in Figure 2, which shows 1 H NMR data from two cell extracts of identical cells
grown in different media. The 1 H NMR spectrum highlights a similar profile for both extracts but cellular histidine
levels are perturbed between the two samples. This demonstrates the power and detail available from NMR-based
metabolomics despite the complexity of the spectra obtained.
Another powerful application of 1 H NMR metabolic profiling is in bioprocessing. An approach called Fermen-
tanomics [9], which studies mammalian cell culture media using NMR, has been shown to optimise amino acid
supplementation in order to achieve optimal antibody production [10]. Another profiling study was used to monitor
both cell extracts and media during cold-shock and recovery of adherent (CHOK1) and suspension (CHOS) Chinese
hamster ovary cell lines to gather information on metabolic load and production [11].
In all metabolic profile studies, it is crucial to identify ‘significant’ metabolic changes when observing profiles;
careful choice of repeat experiments and controls are necessary to classify differences as significant. To maximise re-
search impact, many studies scrutinise the spectral profiles and describe specific metabolite changes in a process that
requires assignment of specific NMR peaks to individual metabolites. As a result, many studies use explicit identifica-
tion of metabolites through additional NMR methods; typically, 2D analytical approaches that separate peak overlap
and aid identification. Alternatively, metabolites can be identified using online databases or software and an overview
of such tools was published by Ellinger and co-workers in 2013 [12].
1
H NMR profile spectra can be cross-referenced with NMR spectra obtained from separated metabolites or even by
spiking metabolites into the extract. For separated metabolites, it is best to follow an established protocol such as that
published for the Birmingham Metabolite Library [13]. Metabolic profiling also has the advantage of being able to
measure the concentration of each metabolite by adding a chemical standard that is present in a known concentration.
This is normally achieved by adding the sodium salt of either 3-(trimethylsilyl)propionic acid (TSP) or 4,4-dimethyl-
4-silapentane-1-sulfonic acid (DSS) at a concentration between 0.01–0.05 mM. Both TSP and DSS provide a strong,
isolated peak at 0 ppm corresponding to nine protons; TSP is labelled in Figure 1. TSP and DSS enable quantitation


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Histidine

(A)

(B)

10.0 9.0 8.0 7.0 6.0 5.0

1
H chemical shift (ppm)

Figure 2. Expanded regions of the 1 H NMR spectra of cell extracts from a single yeast cell line grown in cell culture media
recipes (A and B).
Spectra are comparable but histidine peak intensity changes (↓) confirm elevated levels of this amino acid in the cell lysis extract grown in
media (B). Histidine levels were comparable in both recipes, therefore, each cell media must act differently on histidine biosynthesis in the
cell. Data acquired at 25◦ C using a 14.1 tesla (600 MHz 1 H) Bruker AV3 NMR spectrometer with QCI-F cryoprobe.

because metabolite peaks in standard 1D 1 H NMR spectra have peak areas proportional to their concentrations in
the majority of cases. However, quantitative NMR requires the spectrometer to be set-up correctly and particular
attention must be paid to water suppression, data points/acquisition time and relaxation delay. Water suppression
is a particular issue because pure water contains molecules at a concentration of 55 M at 25◦ C and even in a buffer,
millimolar to micromolar concentrations of metabolites are dwarfed by the 1 H water signal. NMR water suppression is
a complex methodology beyond the scope of this review but several very efficient approaches exist that are reviewed
elsewhere [14]. In our experience, water suppression is best achieved using presaturation when metabolite signals
exist in close spectral proximity to the water 1 H NMR resonance, but labile protons are also ‘at risk’ of attenuation
using this technique. Other suppression methods also exist, such as WaterGATE and excitation sculpting, but their
binomial approach to water suppression can attenuate metabolite signals close to the water resonance, thus obscuring
potentially important species from the subsequent analysis.
As with all analytical techniques, sample preparation is important, and solutions of metabolites have to be extracted
or purified with care to prevent the unwanted introduction of false-positive information. Samples often need to be
treated in such a way as to prevent further metabolic processes that would otherwise interfere with the subsequent
analyses, be it through the addition of antimicrobial agents and/or snap freezing and storage at -80◦ C until immedi-
ately prior to data acquisition [3]. It is important that metabolic differences observed by NMR are due to biological
processes and not, for example, due to differences in cell number, cell volume or quantity of harvested growth media.
In adiition, be aware there are other variables including the method of metabolite extraction and the need to stan-
dardise pH. Many approaches use organic extractions that have been shown to be robust and convenient, whereby
the organic extraction solvent is removed under vacuum and the sample resuspended in H2 O or D2 O prior to NMR
analysis, although additional freeze-drying processes may be used. Lin and collaborators published a study evaluat-
ing metabolite extractions and concluded that methanol/chloroform/water extraction was the preferred method [15].
Should the sample be contaminated with large molecules (e.g. proteins or lipids) NMR methods can be employed to
reduce or remove peaks from such molecules by spectral editing of their fast transverse relaxation rates. Such methods
utilise a ‘spin-lock’ or Carr–Purcell–Meiboom–Gill (CPMG) pulse train within the experiment and can be set-up by
your collaborating spectroscopist.

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Pathway monitoring and metabolic fate

Where a metabolic pathway or specific metabolite is the focus of a study, NMR-based metabolic profiling can still be
utilised but the resultant spectrum is interrogated for the identification and quantitation of specific resonances be-
longing to the metabolite(s) of interest. Spectra automatically include ‘control’ metabolites across the complex profile
to support reproducibility, but these peaks also create overlap and reduce the ability to identify and quantify metabo-
lite peaks in highly populated regions of the spectrum. This ‘dynamic range’ issue with 1 H NMR metabolic profiling
can be circumvented by using 2D NMR methods such as J-resolved (J-res), total-correlation spectroscopy (TOCSY)
or 13 C,1 H-heteronuclear single quantum correlation (HSQC) experiments to reduce signal overlap. However, quanti-
fying metabolites from 2D NMR spectra provides new challenges, as peak intensities in many 2D NMR experiments
are influenced by spin-spin (J) coupling as well as concentration. The process of monitoring specific metabolites is
considered a ‘targeted’ approach and has been successfully applied across medical metabolomics applications such
as the deliberate spiking of blood plasma with 32 specific metabolites to provide phenotyping in breast cancer [16].
However, this should not be confused with a methodological ‘targeted approach’, presented by Weljie and co-workers
[17], where individual NMR resonances are mathematically modelled from pure compound spectra. This method
forms the basis of operation within the software package Chenomx and is commonly applied to metabolic analysis
such as the recent study of the mechanisms of action of antibiotics through NMR metabolomics [18]. This targeted
approach is invaluable in the study of complex biofluids, such as urine, which was shown in a quantitative metabolic
study of human urine using both 1 H and 13 C NMR [19].
Alternatively, specific metabolites may be followed using tracers, whereby a metabolic precursor is introduced and
its fate observed at time points following induction. This is widely described as metabolic flux analysis (MFA) and
typical precursors used include glucose and pyruvate. These molecules are ‘tracers’ because they are 13 C enriched and
their fate is therefore monitored using 13 C NMR. This approach is possible because the natural abundance of the 13 C
isotope is only 1.07% in non-enriched metabolites but is >90% in enriched glucose. As a result, metabolites originat-
ing from 13 C glucose have significantly larger signals in 13 C NMR, but this approach has its limits because as time
progresses the 13 C labelled carbon atoms from the tracer molecule become diluted and move down a multitude of
metabolic pathways. However, careful experiment design can yield amazing results and the strength of this approach
is demonstrated when specific carbon atoms in a tracer molecule are labelled with 13 C, which can be used to iden-
tify where metabolites separate down different pathway branches. Examples include the study of astrocytes in brain
metabolism [20] as well as microbiological studies of CO2 effects on anaerobic succinate production [21]. Although
details of experimental 13 C flux analysis labelling have been published [22,23] they have been typically geared to-
ward gas chromatography-mass spectrometry (GC-MS) studies to date. Sample preparation for flux analysis typically
mirrors that for metabolic profiling.

Specific metabolite and pathway intermediate identification

Nuclear magnetic resonance is pivotal in deducing the molecular make-up of a new metabolic intermediate as well
as establishing new theories regarding the enzymatic processes by which a pathway proceeds. Here, it is critical to
isolate a pure specific metabolic component in sufficient quantities for analysis. New pathway intermediate infor-
mation is useful for future metabolic studies, designing competitive inhibitors and delivering enzymatic knowledge
for targeted drug discovery. Novel structural information can either be deduced through individual studies of each
pathway intermediate, or via the use of specific isotopic labelling to follow the metabolic pathway and register the
chemistries involved. Whatever approach is used, the process is both time- and cost-intensive due to the need to
produce milligram quantities of relatively pure and potentially chemically fragile material that may require isotopic
enrichment.
Examples of pathway intermediate analysis using NMR can be found within recent studies on vitamin B12 (cobal-
amin) biosynthesis. Cobalamin is a complex biomolecule with a core tetrapyrrole structural unit which is only pro-
duced by bacteria and archaea despite being essential for higher order animals. It is a water-soluble vitamin with
a multitude of essential roles in nature including the correct functioning of brain and nervous systems as well as
the formation of red blood cells, and is of nutritional, biomedical and pharmaceutical importance. Both aerobic and
anaerobic metabolic pathways exist in vitamin B12 biosynthesis but the anaerobic pathway has always been perplexing
because the isolation and identification of intermediates is challenging. However, selective overproduction of specific
enzymes enabled the isolation and NMR analytical identification of all metabolic pathway intermediates between
uroporphyrinogen III and cobyrinic acid [9]. This elegant approach utilises an ‘enzyme-trap’ to isolate intermediates
[24] and has also been used to study processes of chemical interest including tetrapyrrole ring contraction [25] and
the biosynthesis of heme and heme-d1 [26]. These projects utilised an explicit suite of NMR experiments to enable


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identification of each macrocyclic pathway intermediate and a selection of such experiments is shown in Figure 3 for
vitamin B12. Of particular interest are 2D experiments such as 1 H,1 H-TOCSY (Figure 3B), 1 H,1 H-NOESY (Figure
3C), 13 C,1 H-HSQC (Figure 3D) and 13 C,1 H-HMBC (Figure 3E) which provide molecular connectivity and through
space proximity information. Although these experiments appear complex, the role of 2D NMR is to simplify data
and help identify the molecule in question. 1 H,1 H-TOCSY provides information that links hydrogen atoms separated
by 3-bonds or less in the molecule, 13 C,1 H-HSQC and HMBC enable carbon atoms to be linked to hydrogen atoms
and 1 H,1 H-NOESY links hydrogen atoms close in space. The combination of all of these datasets is immensely pow-
erful in studies of metabolites as these spectra separate a crowded 1D 1 H NMR spectrum into a second dimension,
thus providing a unique fingerprint for each pathway intermediate and greatly aiding the assignment process. Further
understanding of these experiments can be gained from textbooks and we particularly recommend Claridge’s text for
both chemists and biochemists [27].
No isotopic enrichment was used in the experiments shown in Figure 3 and all of the 13 C NMR spectra were
obtained using the natural abundance of this isotope. This was possible by using a 14.1 tesla (600 MHz 1 H NMR)
QCI-F cryoprobe to provide a much-needed boost in sensitivity where only 0.5–2.5 mg of the pathway intermediate
sample was available, which was dissolved in 0.6 mL of solvent. Also, certain intermediates were air sensitive and
Septum Screw-Cap or Schlenk Line NMR tubes were used with the sample sealed under argon or nitrogen gas.
A cryogenically cooled probehead (cryoprobe) works well to improve sensitivity for pathway intermediate appli-
cations and could be considered an equally useful resource for signal boosting in metabolic profiling. However, room
temperature NMR probeheads are often preferred when reproducible precision is paramount because cryoprobes
can introduce minute variations in chemical shift from sample to sample. Such reproducibility in precision is not as
critical for the identification of intermediates when NMR is employed in tandem with biochemistry and molecular
biology to provide a systems approach to solving the fundamental scientific questions. Such research requires careful
planning, resources and a high-quality interdisciplinary team to maximise impact and success.

Multivariate analysis
Nuclear magnetic resonance-based metabolic data comes in many forms. Specific metabolite and 13 C flux investi-
gations generally utilise analytical organic chemistry NMR procedures that are described in detail within research
texts [27,28]. Data interpretation of metabolic profiles was previously outlined with respect to using databases and
reference spectra to identify metabolites but this complex data can also be statistically analysed for confidence and
significance. The most common approach is to use multivariate statistics, which embraces the complexity of data
where more than a single outcome variable exists. Many researchers opt for the branch of multivariate statistics called
principal component analysis (PCA) that analyses the data and then creates a different group of orthogonal variables
that contain identical overall information as the original set. Unfortunately, explaining PCA operation is beyond the
scope of this review but there are many resources available to describe it, e.g. see reference [29]. Suffice to say, PCA has
the ability to expose the internal data structure in a manner that best explains variance in the data. PCA manipulated
data are usually shown graphically using two or three orthogonal principal component axes with the data mapped
upon it. Data with common principle components tend to form clusters such that if any multivariable differences
exist, these will form a separate cluster. As a result, PCA is a way of ‘seeing the wood for the trees’ when the raw
metabolic spectrum data are complex. However, a word of warning: clustering in PCA needs to be focused for it to be
meaningful and thus concomitant datasets must have low variance. This is the reason that sample preparation must
be tightly monitored and the spectrometer set-up identically for all data acquisitions, thus ensuring that meaningful
results are obtained by minimising variance from data acquisition which drastically reduces the effectiveness of PCA
and its degree of significance. Loss of variance within a known cluster is most probably due to either chemical shift or
concentration variance. The latter is most probably due to poor repetitive sample preparation whereas the former is
influenced by sample pH differences or inconsistent spectrometer operation. Chemical shift variance between equiv-
alent datasets is often higher, and water suppression more difficult, when using cryoprobes which is why there is a
call to return to room temperature probeheads for the next generation metabolism NMR spectrometers. In reality,
cryoprobes can offer an excellent sensitivity boost and should not be completely discouraged from metabolic profile
studies (see Figures 1, 2 and 3).
As an alternate approach to PCA, some researchers opt for partial least squares discriminant analysis (PLS-DA)
that produces a linear regression model by projecting predicted variables and observed variables into a ‘new space’.
An excellent review on PLS-DA was recently published by Gromski and colleagues [30], describing a method that
lends itself to NMR metabolomics by targeted analysis. This paper also discusses the relative ease of PLS-DA due

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(A)

8.0 6.0 4.0 2.0

1
H chemical shift (ppm)

(B) (C)

2 2

H chemical shift (ppm)

H chemical shift (ppm)

4 4

6 6

1
1

8 8

8 6 4 2 8 6 4 2
1 1
H chemical shift (ppm) H chemical shift (ppm)

0 0
(D) (E)
20
50
C chemical shift (ppm)
C chemical shift (ppm)

40

60
100
80

100
150
120
13
13

140
200
8 6 4 2 8 6 4 2
1
1
H chemical shift (ppm) H chemical shift (ppm)

Figure 3. A selection of vitamin B12 NMR spectra used in pathway intermediate identification:
(A) 1 H 1D, (B) 1 H,1 H-TOCSY, (C) 1 H,1 H-NOESY, (D) 13
C,1 H-HSQC and (E) 13
C,1 H-HMBC are shown for 2 mg of vitamin B12 dissolved in
◦ 1
D2 O. All data were acquired at 25 C using a 14.1 tesla (600 MHz H) Bruker AV3 NMR spectrometer with QCI-F cryoprobe. These datasets
also illustrate that the 13 C chemical shift range is larger and distinct from 1 H.

to the wealth of available software, but equally warns against the pitfalls of using such approaches in an unguided
manner. On that note, it is also worth reading Broadhurst and Kell’s article which highlights that many metabolomics
statistical approaches are never described in sufficient detail or suitably validated [31].


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Challenges and the future of metabolism

studies using NMR spectroscopy
The future for NMR-based metabolomics beyond clinical applications looks very bright whereby the use of such
methodologies will enrich multi-omics research projects. Metabolic responses in biological systems are more focused
and will suffer less from the high workflows that hamper clinical metabolomics. As the application of NMR-based
metabolic studies expands through the biosciences, sample preparation, study design, interpretation and data analysis
will improve. This is before new NMR methodologies will assist in answering specific questions; here we predict that
NMR will move beyond 1 H, 13 C (and 31 P) as primary target nuclei and begin to use more exotic species including
non-biochemical elements such as 19 F that have sensitivity approaching 1 H whilst providing a significant leap in data
simplification. There is also additional scope for technological advancements in NMR for metabolomics through re-
ducing chemical shift variance and improved water-suppression from cryoprobes, including the use of liquid nitrogen
cooled probeheads.

Software and online resources

Databases that include NMR data are available from the Metabolomics Centre in Alberta, Canada
(http://www.metabolomicscentre.ca/software). These currently cover the metabolomes of human, bovine, E.
coli, S. cerevisiae as well as housing specialist repositories for drug bank, toxin and target, small molecule pathway,
cerebral spinal fluid and cybercell. This website also has links to software including Metabominer that can be used
to assign complex 2D NMR spectra [32].
The BioMagResBank (http://www.bmrb.wisc.edu) [33], The Human Metabolome Database (www.hmdb.ca) [34]
and the Birmingham Metabolite Library (http://www.bml-nmr.org/) [13] have repositories of data for metabolites
and other biological molecules.
A list of certain metabolomics software for NMR and MS can be found at http://metabolomicssociety.org/
resources/metabolomics-software.
Chenomx commercial software (www.chenomx.com) is widely utilised and enables uploading NMR spectra for
referencing and quantitative analysis. It also includes an expandable database of metabolites.
Multivariate statistical packages come in a variety of forms but many groups use SIMCA (http://umetrics.com/
products/simca) or AMIX (https://www.bruker.com/products/mr/nmr/nmr-software/software/amix/overview.html).
For further details of software and online resources, see reference [12].

Summary
• NMR spectroscopy provides a useful resourceful tool in the study of cellular metabolic processes through
global profiling, specific monitoring and pathway intermediate identification.
• Cells can be monitored for cellular responses to their environment, nutrition, infection, or physical, chem-
ical and biochemical stresses.
• Metabolic variations can be monitored between wild-type and mutant strains to link the metabolome to
genome and proteome.
• Initial investigations are relatively simple to test, providing access is available to a NMR spectrometer and
collaborative technical spectroscopist.
• Sample preparation and data analyses do need careful consideration to deliver robust results for a rigorous
study. This should not be seen as a barrier and there are many tools available to assist in the data analysis
and metabolome mining process.

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Abbreviations
B12, vitamin B12, cobalamin; CHO, Chinese hamster ovary; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; HMBC, heteronu-
clear multiple bond correlation; HSQC, heteronuclear single quantum correlation; MFA, metabolic flux analysis; MHz, megahertz
= 1 × 106 Hz; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PCA, principal component
analysis; PLS-DA, partial least squares discriminant analysis; QCI-F, Quadruple resonance Cryoprobe with Inverse detection and
Fluorine; TOCSY, total correlation spectroscopy; TSP, 3-(trimethylsilyl)propionate; WaterGATE, Water suppression by GrAdient
Tailored Excitation.

Competing Interests
The authors confirm they have no competing interests in relation to this manuscript.

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