Application Note #MSI-02-parti 1
Metabolomics and Metabolite Profiling using Mass Spectrometry Imaging (MSI): A New Biochemical
Tool for Drug Discovery
High resolution mass spectrometry imaging has been used to study endogenous metabolites
from biological tissues. The localization and the identification of small molecules involved in the
main metabolic pathways can be achieved thanks to a FTICR mass spectrometer and a MALDI
ionization source. It allows the possible discovery of new therapeutic targets, the obtention of
valuable pharmacodynamics data or the characterization of most of the metabolism pathways in
animal models.
IntroductionIntroduction
By definition, metabolites are intermediates
and products from different metabolisms. More com-
monly, they are small compounds found in organisms
and have an important role in cells life and survival.
Each of them can have a specific function and repre-
sents a marker of a biochemical process. They can be
nucleotides, amino or organic acids, lipids... Their
identification permits the deconvolution of major
biological pathways (TCA or Urea Cycle, Purine
metabolism, Glycolysis…) and the better understan-
ding of pathologies mechanism (Cardiovascular
disease, Diabetes, Cancer, Inflammatory diseases…).
Different techniques such as Nuclear
Magnetic Resonance (NMR) or Mass Spectrometry
(MS) can be used to characterize these molecules.
Fourier Transform Ion Cyclotron Resonance Mass
Spectrometry (FTICR-MS) is commonly chosen to
study complex biological mixtures thanks to unmat-
study complex biological mixtures thanks to unmat-
ched mass resolution, high mass accuracy and structu-
ral characterization. Therefore, FTICR is the instru-
ment of choice for metabolite profiling in tissue in
combination with MALDI ionization source[1]. The
application of Mass Spectrometry Imaging (MSI) in
the study of endogenous metabolites from biological
tissues is a quite recent but promising technique which
offers to simultaneously monitor several compounds
(drug and metabolites) with spatiotemporal informa-
tion about molecular behavior[2,3]. In this respect, it
can be a valuable tool for pharmacodynamics (the
study of biochemical and physiological effects of
drugs on organism) to understand drug efficacy and
potential toxicity in drug preclinical development.
High resolution mass spectrometry imaging has been used to study endogenous metabolites
1
Representation of main metabolites from different tissues (rat kidney and brain) involved in Krebs cycle (Citrate cycle and
Glycolysis) using MSI ((negative mode, 110 µm of lateral resolution)
Distribution of ribonucleotides in different organs using MSI (negative mode, 110 µm of lateral resolution)Figure 1 DiFigure 1Figure 1
Figure 2 Re
Glyc
Figure 2
IMP ([M-H]-; m/z 347.0396) GMP ([M-H]-; m/z 362.0506) AMP ([M-H]-; m/z 346.0575) H&E Staining
Kidney
Liver
Blood vessel
Glisson's
sheath
Brain
Medulla
Cortex
Corpus
Callusum
Cortex
Calyx
2
Brain, Liver and kidney tissue sections from
control rat were carried out with a Microm cryostat
HM560 (Thermo Scientific, USA), at 10 µm
thickness. All sections were mounted on conductive
ITO glass slides, and then dried. The matrix was
adapted to negative ionization polarities; the 9
amino-acridine (9AA) was choice and applied using
SunCollect Automated Sprayer (SunChrome,
Friedrichsdorf, Germany).
MS images were acquired with a SolariX
7.0T MALDI-FTICR mass spectrometer (Bruker
Daltonics, Bremen, Germany) equipped with a
SmartBeam II laser used at a repetition rate of 1000
Hz. All instrumental parameters were optimized
before the imaging experiment on adjacent tissue
sections. Negative mass spectra were acquired within
the 100- to 1200-m/z range. The mass spectrometer
was operated in the fullscan mode (with on-line data
reduction and the accumulation during detection
mode) and the mass spectrum obtained for each
image position corresponds to the averaged mass
spectra of 500 consecutive laser shots at the same
location. MALDI Images were performed on the
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10000 voxels depending of the tissue dimension).
MS Images were visualized using Quantinetix
software (ImaBiotech, France). Cryosections of
tissue were stained with hematoxylin and eosin
(H&E) solution after MSI analyses in order to
localize fine histological structures.
control rat were c
HM560 (Thermo
thickness. All sec
ITO gl s slides
ResultsResults
nucleic acids (DNA or RNA) but also in the entire
metabolism. These nucleotides consist of a phosphate
group, the sugar ribose, and the nucleobase, the three
metabolites chosen in the present study were:
- AMP or Adenosine monophosphate (m/z
346.0575); AMP could be produced during ATP
synthesis using adenylate enzyme by combining two
ADP molecules. Energy-state of cells or tissues which
control the entire metabolism can be directly assessed
using these metabolites.
- GMP or Guanosine monophosphate (m/z
362.0506); GMP is essential in the activation of G
proteins which is involved in signal transduction.
- IMP or Inosinic acid or inosine mono-
phosphate (m/z 347.0396); IMP has a central
position in Purine Metabolism, (the first nucleotide
formed during the synthesis of purine). IMP act as the
common intermediate of AMP/GMP synthesis.
AMP, GMP and IMP are linked in purine metabolism
which maintains a desired and constant composition
of the nucleotide pool of the organism. Their triphos-
phate forms (also detected using MSI, data not
shown) are the substrates of cyclic nucleotides
(cAMP, cGMP,…) which are secondary messenger
for a large numbers of significant reaction in the orga-
nism (Kinase protein activation, cellular signals
induction,…). All these molecules have been charac-
terized by tandem mass spectrometry (MS/MS) expe-
riment to validate their identification directly in
tissue. The method applied to fragment these species
was the collision induced dissociation abbreviated
“CID”. These ions have specific fragmentation
pattern which exhibit approximately the same daugh-
ter ions especially the phosphate moiety at m/z
94.9696. We can observe that IMP is mainly distribu-
ted in medulla region of the kidney or in the Glisson's
sheath (GS) of the liver but is quite spread over the
entire brain with some variation. Its specific localiza-
tion in kidney and liver (two organs with a high meta-
bolism activity) reflects the importance of IMP in the
metabolic pathways. In this case, MSI gives precise
data that usual analytical techniques without spatial
information couldn’t provide. AMP and GMP are also
partly localized in the medulla or GS region. These
metabolites are closely related in biological pathways
as well as within tissue. In addition, AMP is concen-
trated through the outer structure of the brain; in
Endogenous metabolites are involved in
different diseases as biomarkers of a pathology or
readout molecules used to ensure the efficacy of a
treatment. MSI gives access to a wide range of meta-
bolites depending of ionization properties, small
molecules (AMP or ATP) or lipids
(phosphatydilcholine, ceramides, triglycerides…)
are easily detected by mass spectrometry. As
example, we present here a study of nucleotides in
tissue which play an essential role in the organism
and disease mechanism (Lesch-Nyhan syndrome,
xanthinurie or rena llithiase).
The Figure 1 shows molecular images of
specific ribonucleotides observed in tissue section
using MSI. They are involved in the synthesis of
ell as within tissue. In addition, AMP is concen
through the outer structure of the brain; in
Experimental
ver and kidn
carried out w
Brain, Live
control rat were c
mentalExperime
High resolution mass spectrometry imaging was successfully applied to study in-situ metabolism and to
characterize specific biological pathways.With this techniques, hundreds endogenous metabolites from different
classes could be monitored simultaneously in the same experiment and could give, at the same time, their precise
localization. Also at ImaBiotech, we have generated a database of a few thousands of endogenous metabolites to
accelerate the metabolite research in biological samples. In conclusion, high resolution MSI might become a tool
of choice for better understanding of the metabolic pathways in biological tissues and for pharmacodynamics
studies, for biomarker or therapeutic target discovery.
Conclusion
High resolution m H
ConclusionCo
AdvantagesAdvantages
Discover new therapeutic target
Obtain valuable pharmacodynamics data
Correlate Drug Distribution and readout
variation
Characterize the metabolism of animal
model
Thanks our new service “ImaMet” combining Metabolomics and MSI, ImaBiotech
provides valuable data for drug discovery development by offering new insight into
biochemical process, biomarkers, drug efficacy or toxicity.
contrast GMP has a homogenous distribution in this
tissue. One example of application of our approach
could be the study of the modulation of purine meta-
bolism by an inhibitor used to prevent autoimmune
disease (rheumatoid arthritis, Crohn's disease, ulcera-
tive colitis…). It can be evaluated by monitoring the
associated ribonucleotides in tissue while taking into
account their specific localization. The study of these
molecules which have heterogeneous distribution
within tissue highlights the unique capability of MSI
and its usefulness for readout study or biological
model evaluation.
The molecular image of several metabolites
of Krebs cycle (and glycolysis) in two tissue type
sections is displayed on the figure 2. The Krebs cycle
also known as the tricarboxylic acid cycle (TCA) is
involved in the central metabolism of all organisms
and has a direct importance for all disease processes.
It permits the synthesis of metabolites used in the
anabolism, for example NADH, FADH2 or ATP,
during aerobic metabolism. The figure 2 shows one,
non-exhaustive, example of correlation between
metabolic pathways and molecular imaging of diffe-
rent species. We are able to detect the Glucose
6-Phosphate (m/z 259.0224) which is implicated in
glycolysis that converts glucose to pyruvate. Some
intermediate, as the glycerol 3-phosphate (m/z
171.0064), can also be monitored in this pathway to
have complementary data about metabolism efficacy.
The glycolysis plays an important role in the activa-
tion of the Krebs cycle that is due to Acetyl-CoA or
oxaloacetate production. Its deregulation could be
involved or associated to disease development
(Cancer, Alzeihmer disease…). We can follow the
Krebs cycle operating thanks to the monitoring of the
malate (m/z 133.0320) and the citrate (m/z 191.0620)
molecules. In fact, these two metabolites can repre-
sent some biomarkers of the beginning and the end of
Krebs cycle process. Moreover, the Glutamate (m/z)
and the aspartate (m/z) can also be used as indirect
indicator of oxaloacetate and !-ketoglutarate metabo-
lism. The correlation of the metabolites distribution in
tissue with their role in metabolism may provide a
new insight into the understanding of metabolic dyna-
mics.
BenefitsBenefits
Save time
Reduce costs
Accelerate research
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AuthorsAuthors
Hamm Grégory
Porreaux Lucie
Stauber Jonathan
MS Imaging Department | 885 ave. Eugène Avinée - 59120 Loos - France | +33 (0) 970 440 008 | [email protected]
KeywordsKeywords
Metabolomics
Metabolite
Biological pathways
References
1. Hamm, G., et al., In-situ identification and imaging of metabolites using high resolution mass spectrometry,
in SFSM, Orléans (2012).
2. Daisuke Miura et al., Ultrahighly Sensitive in Situ Metabolomic Imaging for Visualizing Spatiotemporal
Metabolic Behaviors. Anal. Chem. 82, 9789–9796 (2010).
3. Han, J., et al., Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron
resonance mass spectrometry. Metabolomics 4(2): p. 128-140 (2008)
© 2013 ImaBiotech™
ncesReferenc
al., In-situ1. Hamm, G., et al
Small molecule
MALDI-FTICR-MS
Mass Spectrometry Imaging
Jean Viala
Charlène Granier
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