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

!"##$%$&'()%*+&,(+'(+(,-+'"+.(%$,)./'")&()#(001(23(45(

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

4

4

AuthorsAuthors

Hamm Grégory

Porreaux Lucie

Stauber Jonathan

MS Imaging Department | 885 ave. Eugène Avinée - 59120 Loos - France | +33 (0) 970 440 008 | contact@imabiotech.com

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

5