LOCALISATION OF ADENINE NUCLEOTIDES IN MOUSE BRAIN USING ION MOBILITY ENABLED MALDI IMAGING
Eleanor Q. Blatherwick, Bruno G. Frenguelli, James H. Scrivens
University of Warwick, School of Life Sciences, Coventry, UK.
OVERVIEW
Aims
 To develop a sensitive and selective method to successfully identify and localise adenine nucleotides in
mouse brain sections using MALDI imaging.
 Use these methods to compare the intensity ratios and distribution of adenine nucleotides between
control and stressed samples.
Methods
 MALDI Synapt G2 travelling wave ion mobility mass spectrometer (Waters).
Ion mobility separation prior to MS/MS fragmentation and mass spectrometry detection (IMMS).
Results
 Combining ion mobility separation with MS/MS fragmentation provides a selective and reproducible
method for the identification and localisation of ATP, ADP and AMP in mouse brain.
 Using this selective method, ion distribution images of adenine nucleotides for control brain sections
were produced.
BACKGROUND
MATERIALS AND METHODS
Sample Preparation
• Wild type mice were sacrificed by cervical dislocation, followed by decapitation. Control brains were
quickly dissected and snap-frozen in liquid nitrogen. Stressed brains were left at room temperature for 10
minutes following dissection and prior to snap-freezing.
• Brain sections were prepared at 12 µm and thaw-mounted onto glass slides for mass spectrometry
analysis.
• Frozen tissue sections were washed for 1 min in ice-cold 70 % and 95 % ethanol sequentially, before being
brought to room temperature in a vacuum dessicator.
• 10 mgml-1 9-aminoacridine hydrochloride (9-AA) matrix in 70 % ethanol with 0.1 % TFA was applied using
an airbrush for MALDI imaging experiments.
• After imaging experiments, matrix was washed off the tissue sections and H&E staining was performed.
Nucleotide
Precursor ion (m/z)
ATP
506
ADP
426
AMP
346
Intensity Ratios
To calculate relative intensity ratios of ATP:ADP:AMP in brain sections, regions of identical area were
sampled by targeted MALDI-mobility-MS/MS imaging for each of the adenine nucleotides in four brain
sections. Mobility extracted MS/MS spectra were combined for the whole region and the intensity of the
two most intense product ions summed for each nucleotide. These were then used to calculate the
percentage ratio of ATP, ADP and AMP in each brain section. The average percentage ratio and standard
deviation across the brains were calculated for control and stressed brain sections.
RESULTS
MALDI Imaging
Localisation of Adenine Nucleotides in Tissue
MS/MS fragmentation and IMS separation methods were optimised using adenine nucleotide standards to
determine the most selective method for detection of ATP, ADP and AMP, both on and off tissue.
Identification of Adenine Nucleotides in Tissue
The addition of an ion mobility separation step following MS/MS fragmentation allows for the selective
localisation and identification of all three adenine nucleotides in tissue.
All three adenine nucleotides were successfully identified and localised in control tissue using the MALDImobility-MS/MS method described. The mobility extracted MS/MS spectra and arrival time distribution
(ATD) for ATP standard on target and ATP from tissue are shown in Figure 3.
Metabolic Stress
Figure 4. Intensity ratios of ATP:ADP:AMP across four control mouse brain sections with and without an
ethanol wash step during sample preparation. Error bars represent standard deviation across the four brain
sections.
Including the ethanol wash step allowed all three adenine nucleotides to be successfully localised in mouse
brain sections by MALDI imaging. The results are shown in Figure 5.
Figure 6. Intensity ratios of ATP:ADP:AMP across four control and four stressed brain sections. Error bars
represent standard deviation across the four brain sections.
CONCLUSIONS
 Combining ion mobility separation with MS/MS fragmentation provides a selective method for
the identification of ATP, ADP and AMP in mouse brain.
 Inclusion of an ice-cold ethanol wash step prior to defrosting tissue allows for the successful
localisation of all three adenine nucleotides in mouse brain by MALDI imaging.
Information regarding the energetic state of tissue is important in a wide range of experimental studies,
particularly in the study of metabolic stress, such as hypoxia or ischemia. Metabolic stress is known to lead to
the degradation of adenine nucleotides in brain tissue. The energetic state of tissues or cells is often
assessed by determination of adenine nucleotide (ATP, ADP and AMP) levels1. This is commonly achieved
using ultraviolet (UV)-based high-performance liquid chromatography (HPLC), which provides quantitative
data, but does not give specific localisation information.
 This provides a significant step forward from previous work, where localisation images of ATP in
rat brain were not achieved.
 A selective and reproducible method for the identification and localisation of ATP, ADP and AMP
from tissue has been established.
In healthy cells the ratio of adenine nucleotides is maintained with ATP in excess over both ADP and AMP.
During metabolic stress, these ratios change leading to an increase in the levels of AMP present.
 Due to rapid changes in the levels of adenine nucleotides after death, differences cannot
currently be detected between normal and metabolically stressed brain sections.
 Tissue fixation at the point of dissection or at the point of sacrifice needs to be considered to
prevent rapid post-mortem changes in adenine nucleotide levels.
 Mass spectrometry compatible tissue fixation methods will also be evaluated, with the aim to
localise biologically relevant distributions and levels of adenine nucleotides in brain by MALDI
imaging.
 Once a method has been established that provides biologically relevant distributions,
comparisons of these distributions between control and stressed sections can addressed.
Figure 5. MALDI-mobility-MS/MS images of ATP, ADP and AMP product ions from control mouse brain
sections. H&E stained sections are shown below for comparison.
Figure 2. Schematic showing the selective MALDI-mobility-MS/MS experimental method for identification of
adenine nucleotides in mouse brain tissue.
Figure 1. Chemical structures of ATP, ADP and AMP. These adenine nucleotides consist of adenine (blue), a
ribose (black) and a tri-, di- or monophosphate unit (red).
MALDI imaging has been shown to successfully identify ATP, ADP and AMP in rat brain using 9-aminoacridine
as a matrix compound2. Localisation of the adenine nucleotides ADP and AMP was achieved in tissue
sections, but ATP localisation was unsuccessful. This was attributed to the low sensitivity of the experiment.
The area to be imaged was selected using MALDI Imaging Pattern Creator. Data were acquired on a MALDI
Synapt G2 HDMS mass spectrometer operated in HDMS/MS mode over the m/z range 100 – 600 with
MS/MS transitions for each adenine nucleotide as shown in the table. A spatial resolution of 50 µm was
selected and each pixel was sampled for 2 seconds at a laser repetition rate of 200 Hz. After acquisition,
the data was processed and analysed using HDImagingTM software (Waters Corporation) to produce ion
intensity images.
Figure 3. MALDI-mobility-MS/MS spectra and arrival time distributions of ATP standard on target (top,
green) and m/z 506 from brain tissue (bottom, red).
Similar results were achieved for both ADP and AMP from control mouse brain, showing successful
identification of the adenine nucleotides in tissue. Localisation images for all three adenine nucleotides
were produced but were not as expected for control healthy tissue.
The intensity ratio of ATP:ADP:AMP was compared across four control brain sections, revealing lower levels
of ATP than expected. An ice-cold ethanol wash step was included in the sample preparation procedure to
prevent hydrolysis of ATP as the tissue is brought to room temperature from frozen. Figure 4 shows the
improvement in ATP levels by including the ethanol wash.
Comparison of Control & Stressed Brain Sections
Targeted experiments for ATP, ADP and AMP were run across four control and four stressed brain sections.
All tissue sections were subjected to an ice-cold ethanol wash step prior to warming to room temperature.
The average intensity ratios of ATP:ADP:AMP in control and stressed tissue sections are shown in Figure 6.
Figure 6 shows very little difference between the intensity ratio of adenine nucleotides in control and
stressed brain sections. This is unexpected, as ATP levels in control (healthy) tissue should in excess over
both ADP and AMP. In tissue subjected to metabolic stress, the level of ATP should be significantly lower
with AMP in excess.
REFERENCES
1. Zur Nedden, S., Eason, R., Doney, A.S. & Frenguelli, B.G. “An ion-pair reversed-phase HPLC method for
determination of fresh tissue adenine nucleotides avoiding freeze-thaw degradation of ATP” Analytical
Biochemistry, (2009). 388: p108-114.
2. Benabdellah, F., Touboul, D., Brunelle, A. & Laprevote, O. “In Situ Primary Metabolites Localization on a
Rat Brain Section by Chemical Mass Spectrometry Imaging” Analytical Chemistry, (2009). 81: p5557-5560
ACKNOWLEDGEMENTS
I would like to thank the Medical Research Council and AstraZeneca for my PhD project funding.