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This review provides an overview of two complementary approaches to identify biologically active compounds for studies in chemical ecology. The first is activity-guided fractionation and the second is metabolomics, particularly focusing on a new liquid chromatography-mass spectrometry-based method called isotopic ratio outlier analysis. To illustrate examples using these approaches, we review recent experiments using Caenorhabditis elegans and related free-living nematodes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.
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SYMPOSIUM
Metabolomics and Natural-Products Strategies to Study Chemical
Ecology in Nematodes
Arthur S. Edison,
1,
* Chaevien S. Clendinen,* Ramadan Ajredini,* Chris Beecher,*
,†
Francesca V. Ponce* and Gregory S. Stupp
*Department of Biochemistry and Molecular Biology and Southeast Center for Integrated Metabolomics, University of
Florida, Gainesville, FL 32610-0245, USA;
IROA Technologies, Ann Arbor, MI, USA;
The Scripps Research Institute,
Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
From the symposium ‘‘Chemicals that Organize Ecology: Towards a Greater Integration of Chemoreception,
Neuroscience, Organismal Biology, and Chemical Ecology’’ presented at the annual meeting of the Society for Integrative
and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida.
1
E-mail: aedison@ufl.edu
Synopsis This review provides an overview of two complementary approaches to identify biologically active compounds
for studies in chemical ecology. The first is activity-guided fractionation and the second is metabolomics, particularly
focusing on a new liquid chromatography–mass spectrometry-based method called isotopic ratio outlier analysis. To
illustrate examples using these approaches, we review recent experiments using Caenorhabditis elegans and related free-
living nematodes.
Introduction
This review focuses on a variety of analytical tech-
niques and approaches that can be used to solve bi-
ological problems, especially those in chemical
ecology and in behavior. As such, we will summarize
some of the different technologies that are now avail-
able to characterize metabolites and low molecular
weight signaling molecules. Each method has its
own particular strengths and weaknesses, which we
will emphasize by giving specific examples: research
that we, and others, have done over the past decade
on the model organism Caenorhabditis elegans and
related nematodes. Even though it is best known as
a model organism for genetics (Brenner 1974) and
development (Sulston and Horvitz 1977), C. elegans
is also an ideal animal for chemical studies
(Schroeder 2006).
It is worth beginning with a brief discussion
of two areas of chemical science that are particularly
relevant to studies in ecology and behavior. The first
is natural products chemistry, which typically starts
with a complex mixture from an extract of an
organism of interest, partially or fully purifies an ac-
tive compound(s) using activity-guided fractionation
(AGF), and identifies the active compound(s) using
nuclear magnetic resonance (NMR) and/or mass
spectrometry (MS), which often is coupled to
liquid or gas chromatography (LC or GC). The
second is metabolomics, which typically starts
with complex mixtures from two or more groups
that differ by a specific phenotype of interest,
collecting NMR or LC–MS/GC–MS data, and
comparing the datasets statistically to identify bio-
markers that show differences among the groups.
The differences and similarities of natural products
chemistry and metabolomics are summarized in
Fig. 1, which was adapted from a recent review ar-
ticle comparing NMR-based natural products and
metabolomics. The conclusion of that article is that
the two fields are very closely related and, in fact,
are merging in significant ways. Both start with
complex mixtures of small-molecule metabolites,
use the same analytical technologies, and have as a
primary goal the identification of biologically ac-
tive molecules or biomarkers (Robinette et al.
2012). Similar conclusions have been drawn using
LC–MS (Constant and Beecher 1995;Wolfender
et al. 2015).
Integrative and Comparative Biology
Integrative and Comparative Biology, pp. 1–8
doi:10.1093/icb/icv077 Society for Integrative and Comparative Biology
ßThe Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.
0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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The plan of this review is as follows. The first
section focuses on the use of AGF to target specific
compounds of interest. This is a time-honored ap-
proach that is capable of leading to active com-
pounds, even in low quantities. The drawback is
that AGF is also time-consuming and only focuses
on relatively few compounds. We then show how
metabolomics can be used to obtain chemical infor-
mation on a more global level, particularly focusing
on a new LC–MS technique called isotopic ratio out-
lier analysis (IROA). We summarize several different
approaches that are relevant to chemical ecology
studies. Finally, we close with some ideas regarding
strategies for integrating phenotypic data with natu-
ral products and metabolomics to gain new biologi-
cal insight on a systems level.
Activity-guided fractionation
AGF is conceptually simple and extremely useful as a
method for discovering molecules with specific
functions, but the necessary chromatography can be
quite complex. The most important factor in AGF is
the assay to measure the activity, which should be sen-
sitive, easy, fast, and reproducible. These can be based
on chemistry or biochemistry, such as enzymatic activ-
ity, or involve cells or organisms as detectors. A notable
assay used male moths in cages as detectors in the pu-
rification of the active component of the mating pher-
omone, as reviewed by Hummel (1984). A more recent
and high-tech assay used neuronal recordings of a
moth’s antennal lobe combined with GC to identify
complex odors associated with plant–pollinator rela-
tionships (Riffell et al. 2009). Interestingly, male ele-
phants in a zoo were used to monitor the purification
of males’ mating pheromone. We leave it to the imag-
ination of the readers or to the original reference for a
description of this purification (Rasmussen et al. 1997).
As an evolutionary side-note, moths use the same
mating pheromone as a component of their signaling
system (Rasmussen et al. 1997).
Fig. 1 Schematic comparison of two similar strategies to identify active compounds or biomarkers: natural products using activity-
guided fraction on the left and metabolomics on the right. Both approaches use similar technology but different steps to get to the
same endpoint. Steve Robinette made this figure, which was adapted with permission from Robinette et al. (2012).
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We have used male C. elegans as detectors to
purify the first mating pheromone in nematodes
(Srinivasan et al. 2008). Details are provided in the
primary reference, but the general procedure was to
isolate large quantities of exudates from liquid cul-
tures of C. elegans hermaphrodites and test these for
male-specific responses using simple two-spot assays
on agar plates, in which one spot has a buffer control
and the other spot has a test fraction. We then mon-
itor the time spent by males in each spot (Fig. 2).
There are several important considerations when
doing one of these assays. First, male and hermaph-
roditic worms respond to many things, including
bacterial food. Therefore, bacteria must be separated
from the worms to get a specific and meaningful
response. We developed a ‘‘worm water’’ prepara-
tion, in which a synchronized population of her-
maphrodites at a specific developmental stage was
separated from bacteria using a sucrose gradient
(Srinivasan et al. 2008;Kaplan et al. 2011;Choe
et al. 2012). Then, the worms were allowed to sit
in water or a defined buffer, and the supernatant
was collected for bioassays.
When purifying an unknown compound, it is im-
portant to develop a simple but accurate ‘‘account-
ing’’ system. Since by definition, we do not know
what we are looking for before we find it, we can’t
use molarity. When working with worms, we use our
own units called ‘‘worm equivalents’’, for which 1
worm equivalent is defined as the amount of mate-
rial released by one worm in 1 h. Any convenient and
consistent definition will suffice. Using this simple
metric, it is easy to identify synergy or loss of activity
or to detect other confounding factors.
We discovered after our initial study that male
worms were able to respond to as little as 2.5
worm equivalents, which makes biological sense
(Kaplan et al. 2011). It was also humbling to dis-
cover that our NMR spectrometer required about 4
million worm equivalents to detect the same phero-
mone (Fig. 3). This illustrates possibly the most im-
portant advantage of AGF over the metabolomics
experiments described below: with a very sensitive
bioassay like a male worm or moth, it is possible
to detect the important components at concentra-
tions that would be entirely missed with analytical
instruments.
Male worms may be extremely sensitive, but this
can add other potential complications to an AGF.
First, many response curves for pheromones are
bell-shaped, with maximal responses tuned to appro-
priate concentrations and falling off at higher or
lower concentrations, presumably stopping mate-
finding behavior when a mate is located (Fig. 4a).
It is important at the start of a new AGF study to
carefully examine the crude extract (e.g., ‘‘worm
water’’ for many of our studies) for activity at a
wide-range of concentrations (e.g., worm equiva-
lents) to avoid putting too much material onto the
assay. However, this can also backfire, because if
there are different compounds that cause the oppo-
site response or block the primary response, the
starting material may not work. A good example of
teasing apart opposite responses in behavioral assays
was an assay developed by Srinivasan et al. (2012) to
characterize simultaneous attractive and repulsive re-
sponses to a mixture of two related pheromones in
C. elegans. Briefly, three concentric scoring regions
were defined, and the time spent in the center was
compared with the time spent in the outer circles.
One of the major complications of AGF is a syn-
ergetic effect of different metabolites responsible for
Fig. 2 AGF results for the purification of the male-specific mating pheromone from cultures of hermaphroditic Caenorhabditis elegans.
The assay is shown in (a), where about five worms were placed on each of the spots marked with ‘‘x’’ and either buffer control or
hermaphrodite-conditioned media were added to the spots in the center. The worms were videotaped and scored by how long they
stayed in each spot, which is plotted in (b). The data in (b) correspond to material collected from controls (C) and hermaphrodites at
different developmental stages (Egg, L1, L2, L3, D ¼dauer, L4, YA ¼young adult, and A ¼adult). Jagan Srinivasan conducted the assays
and made the figure, which is reproduced with permission from Srinivasan et al. (2008).
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the activity of the crude extract. In our study of the
identification of males’ mating pheromone in C. ele-
gans, we lost all activity at a key step in the fraction-
ation (Srinivasan et al. 2008). The loss of activity
could be caused by several factors, including degra-
dation of the molecule(s), loss of the molecule(s) on
the column, or synergy. Combining all the fractions
and testing for activity, which will return if two or
more compounds are acting synergistically, can rule
out degradation or loss. With synergy, AGF gets
more complicated. The evidence of synergy in that
study is shown in Fig. 4b. Often a good start is to
identify a fraction that is necessary but not sufficient
for activity. This will yield at least one component,
which can then be added to other fractions for ad-
ditional bioassays.
In summary, AGF is a powerful but potentially
very time-consuming way to identify biologically sig-
nificant molecules. It can often reveal activity at con-
centrations that would be too low to measure with
analytical instrumentation, and since it is always di-
rected to the activity of interest, positive results will
Fig. 3 1D
1
H NMR spectrum from our study to isolate and identify a component of the mating pheromone of Caenorhabditis elegans
(Srinivasan et al. 2008). This spectrum was collected using one of the most sensitive NMR probes available (Brey et al. 2006). Because
the pheromone is present in such low concentrations, this spectrum required about 4 million worm equivalents of material. A male
C. elegans can detect as little as 2.5 worm equivalents (Kaplan et al. 2011).
Fig. 4 Dose response curves (a) and synergy (b) of the attraction of male Caenorhabditis elegans to two different mating pheromones.
The assays were as described in Fig. 2. Panel (a) shows that there can be points of maximal attraction and that caution needs to be
used to avoid adding too much material in a bioassay. The blue and red arrows in (a) indicate the concentrations tested in (b), which
shows the results of adding together two pheromones at concentrations that each alone would produce no activity but that together
produce a large response. All of these very interesting biological stories can cause great confusion in an AGF study. Reproduced from
Srinivasan et al. (2008).
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be relevant to the study. Synergy, compounds with
masking or opposing activities, and bell-shaped re-
sponses can all complicate AGF. We have conducted
two large AGF identifications of nematodes’ mating
pheromones, and they both required several years of
work to complete (Srinivasan et al. 2008;Choe et al.
2012); they are definitely not high-throughput!
Metabolomic strategies
The right side of Fig. 1 summarizes a metabolomics
approach to the identification of biomarkers. As
illustrated, the end-point of a metabolomics and
natural-products (AGF) study can be the same, but
the mechanism and steps to get there are quite
different. In metabolomics, the laborious step of
AGF is eliminated. This can be wonderful news to
anyone who has conducted an AGF study. There are
some important differences in terms of overall design
and outcomes of experiments between AGF and
metabolomics.
The first is replicates of samples. Historically, AGF
studies have not focused on obtaining large numbers
of unique biological replicates, but in most cases
many different samples need to be generated over
the course of a study for subsequent steps in a pu-
rification scheme. In contrast, metabolomics is com-
pletely dependent upon statistical analysis. Therefore,
a well-controlled design of the study, including ap-
propriate numbers of replicates, is critical. It is
always important in a metabolomics study to have
discussions with an expert statistician.
Another important difference between AGF and
metabolomics is the point at which analytical sensi-
tivity becomes a major factor. Both approaches use
the same types of MS and NMR instruments, but the
reliance on the instruments is significantly different.
In the example described above, male C. elegans were
able to detect a pheromone produced by about 2.5
worm equivalents (Kaplan et al. 2011), while an out-
standing NMR system (Brey et al. 2006) needed
about 4 million worm equivalents of the same ma-
terial. Based on knowledge gained from subsequent
studies, we estimate that a sensitive LC–MS instru-
ment would need about 10,000 worm equivalents for
reliable detection of the same material (Stupp et al.
2013). The critical point here is that if we were re-
lying entirely on NMR and MS instruments, as in the
case in metabolomics, we would not find an active
compound that is below our detection limits.
However, with AGF, we know something of interest
to the male worms is there, even if we need to pro-
duce a lot of material and concentrate it for NMR
and MS. Thus, with metabolomics it is critical to
always remember that the analytical instrumentation
will define the number and concentrations of metab-
olites detected. Important biologically active com-
pounds at concentrations too low to measure will
be completely invisible in a metabolomics study.
One important distinction to make for metabolo-
mics is ‘‘targeted’’ versus ‘‘untargeted’’ or ‘‘global’’
metabolomics. A full discussion of these is beyond
the scope of this review, but excellent review articles
have been published (Fiehn 2002;Dunn et al. 2011).
In many ways, AGF could be considered a type of
targeted metabolomics, for which the goal is to iden-
tify and quantify a specific compound based on ac-
tivity. Targeted assays generally refer to classes such
as ‘‘amino acids’’, ‘‘organic acids’’, but by using a
biological detector like a male worm, a target class
could also be ‘‘the group of compounds that causes
males to be attracted’’. Global metabolomics has sev-
eral compelling advantages over targeted studies if an
unbiased view of a given system (e.g., hypothesis-
generation) is required. There are several different
types of metabolomics experiments and general
approaches to complex mixtures. We have recently
reviewed and contrasted some of the methods used
in NMR (Robinette et al. 2012). Here, we briefly
describe a new metabolomics experiment that we
think has some particular advantages for studies in
chemical ecology.
As suggested above in the comparison of how
many worm equivalents were needed to characterize
a mating pheromone of C. elegans, NMR is less sen-
sitive than MS. NMR typically requires about 100
nanomoles of material, but significant improvements
with specialized technology can lower that to the just
a few nanomoles (Dalisay et al. 2009;Molinski 2010;
Ramaswamy et al. 2013). The major advantage of
NMR is that it provides atom-specific information
that is necessary for the full identification of an un-
known molecule. NMR is also very reproducible, in
part because the sample is in a tube that is not in
contact with the instrument. Finally, NMR can be
quantitative when the spectroscopist allows for full
relaxation between scans. In contrast, MS is ex-
tremely sensitive, requiring much less material than
NMR. For targeted analyses of known molecules, MS
is generally much more efficient than NMR. On the
other hand, LC–MS data can have many artifacts,
and reproducibility is challenging, in part because
the sample is in contact both with the chromato-
graphic column and the detector in the MS instru-
ment. MS data provide the mass/charge of each
detected ion, and some instruments are capable
tandem MS (MS
n
), which fragments ions that can
be analyzed de novo or matched to databases for
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more definitive identification. Several reviews are
available that expand on these basic introductory
concepts (Katajamaa and Oresic 2007;Moco et al.
2007;Scalbert et al. 2009;Edison and Schroeder
2010;Wolfender et al. 2013).
Isotopic ratio outlier analysis
IROA is a relatively new LC–MS-based experiment
that we have used in experiments on metabolomics
in C. elegans (Stupp et al. 2013). It has been reviewed
(de Jong and Beecher 2012), so here we will focus on
its utility for chemical ecology. In the basic IROA
experiment, two different populations are compared,
one that has been labeled with 5%
13
C and the other
with 95%
13
C.
13
C is a stable isotope that not only is
useful for MS experiments but also is an excellent
NMR nucleus, allowing for some combined analysis.
The basic IROA workflow is shown in Fig. 5. There
are several advantages of IROA for LC–MS metabo-
lomics. First, the patterns of peaks created by the
isotope labeling are easy to detect by a computer,
greatly reducing artifacts. Second, the number of
carbon atoms is known exactly for each MS peak,
allowing for very efficient determination of molecu-
lar formulas. Finally, the relative quantities of the 5%
and 95% channels are easily quantified. The result is
that hundreds to thousands of peaks can be reliably
detected and quantified. It is relatively easy to assign
peaks to a molecular formula, but it is a greater
challenge to figure out the true identity of all the
peaks.
There are many experiments that one could do
with IROA. We will illustrate these with examples
Fig. 5 IROA method: (a) Experimental and control groups of worms are isotopically labeled at 5% or 95%
13
C and grown to the stage
of young adults. The experimental group is split into four replicates and is perturbed, while the control group is not split. After
incubation, the control group is split into four replicates, and each replicate is mixed 1:1 with an experimental replicate (b) for uniform
preparation of samples and for LC–MS analysis. (c) Biological compounds are easily distinguished from artifacts by the recognizable
pattern caused by the isotopic enrichment. (d) Using automated software, the fold-changes for all detected biological compounds can
be determined. Reproduced with permission from Stupp et al. (2013).
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from nematodes, but virtually any system that can be
labeled can be studied. For example, wild isolates of
C. elegans, or other free-living species, could be col-
lected and labeled with 5%
13
C using labeled bacte-
rial food. These could be compared with a common
reference material of the laboratory wild-type (N2)
strain that had been labeled with 95%
13
C. With a
very large batch of reference material, many different
pairwise experiments can be conducted, and differ-
ences between wild isolates and N2 can be deter-
mined. These metabolic differences can then be
compared with behavioral differences to correlate
molecules or pathways with the phenotype.
Another example would be to label a given strain
with both 5% and 95%
13
C and then add an unla-
beled pathogen or other perturbation to the 5%
channel. In this scenario, the global metabolic re-
sponses to the perturbation can be measured.
Finally, in an experiment called ‘‘phenotypic
IROA’’ (de Jong and Beecher 2012), the 5%
13
C
channel can be replaced by natural abundance
13
C,
which simply changes the pattern of isotopic distri-
bution on the
12
C side (blue peaks in Fig. 5). Some
information is lost from the traditional IROA exper-
iment, but with this it is possible to compare differ-
ent species even if some cannot be cultured. For
example, parasitic nematodes are notoriously difficult
to culture, but one could compare these to a refer-
ence 95%
13
C labeled C. elegans. It is important to
note that in this situation, the experiment is more
similar to a targeted study in which the targeted
compounds are all labeled compounds from the ref-
erence strain, because they are the only ones that can
be detected.
Conclusion
We have attempted to illustrate different approaches
that we have used to study chemical ecology in com-
plex systems. The examples of nematodes follow
from our own work and interests, but many different
types of systems can be studied using similar exper-
iments. There is no ‘‘perfect’’ approach, and in prac-
tice a combination of a natural products-based AGF
and metabolomics is perhaps ideal. To find out the
molecular basis of a specific behavior, a good starting
point would be a simple and reliable bioassay for
that behavior, followed by an AGF of the active com-
pound(s). In parallel or sequentially, a metabolomics
experiment could be developed to look more globally
at the problem, either to avoid bias or to capture
other compounds that may be relevant. A nice ex-
ample of a combined approach is illustrated by
the identification of both a male-specific and a
female-specific mating pheromone in Panagrellus
redivivus (Choe et al. 2012). In that study, the
Edison and Sternberg laboratories conducted AGF
on a mixed culture of worms and continued until
gender-specific components were separated chro-
matographically. Two different ascarosides were dis-
covered, ascr#1 that attracts males and dhas#18 that
attracts females. Simultaneously, the Schroeder labo-
ratory conducted a metabolomics-type experiment
that used LC–MS to detect all ascarosides made by
the species. They discovered ascr#1 and dhas#18,
along with several other ascarosides not found in
the AGF study. However, upon testing of the other
ascarosides, none of them had significant activity in
gender-specific attraction, suggesting other unknown
functions (Choe et al. 2012). This study nicely illus-
trates the power both of natural products AGF and
of metabolomics, which often can be used together
to approach a question from two sides of the same
coin (Robinette et al. 2012).
Acknowledgments
A.S.E. would like to dedicate this work to the
memory of Dr Peter Teal, a friend and colleague
who taught AGF to members of the Edison labora-
tory. A.S.E. thanks the SCIB organizers for inviting
him to present some of this work in the 2015 meet-
ing in Palm Beach, FL. The authors thank John
Hildebrand and Jim Tumlinson for their feedback.
Funding
This work was supported by the Southeast Center for
Integrated Metabolomics (NIH 1U24DK097209).
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Arthur S. Edison obtained a B.S. in chemistry from the University of Utah, where he studied monoterpenes isolated from southern Utah sagebrush by NMR. He completed his Ph.D. in biophysics from the University of Wisconsin, Madison, where he developed and applied NMR methods for peptide and protein structural studies under the supervision of John Markley and Frank Weinhold. In 1993, Dr. Edison joined the laboratory of Anthony O. W. Stretton at the University of Wisconsin as a Jane Coffin Childs postdoctoral fellow where he investigated the role of neuropeptides in the nervous system of the parasitic nematode Ascaris suum. He joined the faculty at the University of Florida and the National High Magnetic Field Laboratory in 1996 and is currently the Director of Chemistry & Biology at the NHMFL. Dr. Edison’s current research is in technology development for high-sensitivity NMR and natural product discovery in nematodes and other invertebrates. Dr. Edison is the recipient of the 1997 American Heart Association Robert J. Boucek Award, a CAREER Award from the National Science Foundation in 1999, and, with his postdoctoral scientist Aaron Dossey, the Beal award for the best publication of the year in the Journal of Natural Products in 2007.
Chapter
Nuclear magnetic resonance spectroscopy (NMR) provides a rich source of structural information which when combined with other spectroscopic data allows structural elucidation of complex compounds as well as a universal detection method for compounds of interest in complex mixtures. NMR is a nondestructive technique that facilitates its coupling with other spectroscopic methods such as mass spectrometry. The key to on-line NMR detection is the design of the flow probe. Suitable probes for continuous flow, stop flow, loop collection, solid-phase extraction, and microflow probes are described emphasizing their general applications and limitations. Sensitivity and probe volume are major limitations of NMR coupling to liquid chromatography. Solvent selection for separations needs to consider the unique characteristics of NMR, for example the use of deuterated solvents for proton detection, and methods available for solvent suppression. Practical applications of LC-NMR are presented to illustrate the use of this technique in different areas of analytical chemistry.
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We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass spectrometry-based technique called Isotopic Ratio Outlier Analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95% and 5% 13C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: 1) compounds arising from biosynthesis are easily distinguished from artifacts, 2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, 3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulae, and 4) relative concentrations of all metabolites are easily determined. A heat shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including many compounds from the purine metabolism pathway, which we use to demonstrate the approach. The metabolomic response information for C. elegans provided by IROA may be interpreted in the context of a wealth of genetic and proteomic information available. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline.
Chapter
Genuinely new detection methods like the flame ionization detector (FID)3 for gas chromatography (McWilliams and Dewar, 1958; Harley et al., 1958), mass spectrometry for structure determination (Biemann, 1962), and the use of whole insects (Flaschenträger et al., 1957) or isolated insect antennae (Schneider, 1957; Roelofs, 1984, Chapter 5, this volume) as biological detectors significantly increased the number of techniques available to the pioneers in the field 25 years ago (see Hecker and Butenandt, 1984, Chapter 1, this volume). Equally important and numerous are the examples in which a novel combination of already existing techniques opened up additional analytical avenues. Examples are coupled gas-liquid chromatography-mass spectrometry (GLC- MS) (Gohlke, 1959; Ryhage, 1964), tandem GLC-EAD (Moorhouse et al., 1969; Arn et al., 1975; Beevor et al., 1975), tandem GLC single-cell recordings (Wadhams, 1982; Wadhams, 1984, Chapter 7, this volume; Löfsted et al., 1982; Struble and Arn, Chapter 6, this volume), and the tandem GLC-behavior bioassays (t-GLC-BB) (Table 1).
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With the advent of Electrospray HPLC/MS instruments and a database like NAPRALERT, a quick and easy method for the dereplication of natural products can now be achieved.