Download Small Molecule Drug Imaging of Mouse Tissue by MALDI

Bruker Daltonics
Application Note # MT-93/FTMS-38
Small Molecule Drug Imaging of Mouse Tissue by
MALDI-TOF/TOF Mass Spectrometry and FTMS
Introduction
Matrix Assisted Laser Desorption Ionization (MALDI)
Mass Spectrometry (MS) with a computer-controlled
xy-stage in the ionization source can be used to detect
the presence, approximate amount and location of small
molecules, lipids, peptides and proteins in tissue samples
through in vitro molecular imaging, often referred to as
MALDI imaging. Both MALDI - Time-of-flight/Time-of-flight
(MALDI-TOF/TOF MS) and MALDI - Fourier Transform
Mass Spectrometry (FTMS) were used to detect and image
the location of Erlotinib in various mouse tissues. The
molecular imaging experiments not only indicated where in
the mouse’s body the drug accumulated, but it also gave
insight regarding the different MALDI imaging technologies’
respective capabilities for small molecule drug tissue
imaging.
Erlotinib is used as a cancer fighting agent, and is a human
epidermal growth factor receptor type 1/epidermal tyrosine
kinase inhibitor (1). Erlotinib has a molecular formula of
C22H23N3O4HCL, and a mass of 394 Daltons, after the
replacement of the HCL salt by a hydrogen atom. Erlotinib
inhibits the intracellular phosphorylation of tyrosine kinase
associated with the epidermal growth factor receptor. The
epidermal growth factor receptor is expressed on the cell
surface of normal cells and cancer cells. Erlotinib focuses
on brain tumors and inhibits their growth, thus increasing
cancer patients’ length of time of survival. When used in
studies against a placebo, the one year survival rate of
patients with certain brain cancers increased from 21.5% to
31.2%. According to Kaplan-Meier the chance of surviving
up to 24 months with the brain tumor increases from a 0.1%
to approximately a 20% chance of survival (2) (Figure 1).
Erlotinib is intended to assist other stronger anticancer
drugs as a specialized companion drug that is less likely
to have negative side effects, due to its ability to more
effectively target cancer cells than normal cells. Erlotinib
is also utilized in pre- and post-surgery treatment. If taken
pre-surgery, Erlotinib shrinks tumors, reducing the chance
of regular cells being removed along with the tumor. If taken
post-surgery, Erlotinib can help eliminate remaining tumor
cells (3).
Experimental
Mice were injected with human brain tumor cells, and given
Erlotinib via oral gavage at a concentration of 100 mg/kg.
The mouse was euthanized four hours later. The kidney,
liver and brain were immediately removed from the mouse,
flash frozen in liquid nitrogen, and sliced into frozen tissue
sections with a Microm HM 525 cryostat (Thermo Fisher
Erlotinib increases chance of survival
Fig. 1: Kaplan Survival Survey: The graph shows the probability of
survival over time with a brain tumor when using the drug Erlotinib
against the placebo. Erlotinib allows the average patient to live longer
compared to patients receiving a placebo.
Fig. 2: MALDI-TOF and TOF/TOF Measurements of Erlotinib standard. The top spectrum is of the neat compound measured in MS-mode
showing the parent ion at 394 Daltons. The bottom spectrum shows the neat compound being measured in TOF/TOF-mode with strong
fragment ions at 336 and 278 Daltons which are unique to Erlotinib.
Confirmation spectra of Erlotinib sample and fragment ions
Detection of Erlotinib parent ions in kidney and liver
Fig. 3:
MALDI-TOF Image
of kidney slice
showing Erlotinib
distribution. Many
pixels have a peak
at 394 Daltons and
show traces of the
drug in the mouse
kidney.
Fig. 4:
The MALDI Image
of mouse liver
tissue shows the
presence of Erlotinib
throughout the
tissue sample.
Detection of Erlotinib ion fragments in liver
Fig. 5: The MALDI-TOF/TOF Image of the mouse liver tissue shows the presence of the ion fragments of Erlotinib.
MALDI-FTMS confirmation of results
Fig. 6: MALDI-FTMS Spectrum in Liver Tissue.
The MALDI-FTMS Spectrum extracted from the
MALDI Image shows the Erlotinib molecular ion
peak at 394 Dalton with excellent signal-to-noise
ratio of better than 50:1, which suggests that the
drug could be detected and imaged at even lower
concentrations.
Erlotinib distribution in the liver tissue slice
A ultra-high mass resolution and high mass accuracy
apex®-ultra MALDI-FTMS (Bruker Daltonics, Billerica, MA)
was also used to measure the liver tissue in MS- mode with
the following parameters: the pixel step size was 100um,
the laser spot diameter was 30um, with a total of 800 laser
shots per pixel collected in 200 shot increments.
Results
Fig. 7: MALDI-FTMS Image of Liver Tissue showing Erlotinib
Distribution
Scientific Inc., Waltham, MA). The temperature of the
cryostat was -25° C. The mouse brain, liver and kidney
tissue slices were transferred onto glass Indium Tin Oxide
(ITO) slides and then stored at -70° C.
Prior to MALDI Imaging of the tissue, neat samples of
Erlotinib were measured by MALDI-TOF MS with two
matrices, alpha cyano-4-hydroxycinnamic acid (Sigma
Aldrich, St. Louis, MO) and 2,5-Dihydroxybenzoic acid
(DHB), (Bruker Daltonics, Billerica, MA). The DHB matrix
yielded better results and was therefore used for the
MALDI Imaging experiments.
Multiple layers of matrix were uniformly deposited onto
the tissue samples using the MALDI Imaging automated
preparation system ImagePrep™ (Bruker Daltonics, Billerica,
MA). DHB matrix was dissolved in 60% acetonitrile and
0.2% TFA at a concentration of 10 mg/ml. Three tissue
sections were inserted into either the autoflex™ III MALDITOF/TOF, or the ultraflex™ III MALDI-TOF/TOF (Bruker
Daltonics, Billerica, MA), each equipped with a Scout
MTP™ microtitre plate sized computer-controlled xy sample
stage and a smartbeam™ 200 Hz solid-state laser.
Each tissue section was measured with 100um pixel steps
with a laser spot diameter of 30um. Two hundred laser
shots were accumulated for each pixel. The data was
collected in both MALDI-TOF MS-mode, and MALDI-TOF/
TOF MS/MS-mode.
For confirmation, the neat Erlotinib sample was measured
in MS-mode to confirm the molecular ion of 394.17 Daltons.
The sample was also measured in TOF/TOF mode to
generate fragment ions at 336 and 278 Daltons (Figure
2). Liver, kidney and brain tissue samples were measured
with MALDI-TOF and MALDI-TOF/TOF (Figures 3-5),
while only the liver tissue was measured with MALDIFTMS (Figure 6). Erlotinib was detected in all liver and
kidney measurements but no drug could be detected in
the brain tissue. MALDI-FTMS was used to confirm the
results found in the liver sample. The strongest signal was
obtained from the FTMS measurement as seen in Figure 7.
This is due in part to the higher total number of laser shots
per pixel, as during FTMS analysis we had acquired 4 times
more shots per pixel compared to MALDI-TOF. In addition,
the high mass resolution capabilities of MALDI-FTMS, with
MALDI Imaging mass resolution of >100,000, enable better
discrimination of peak signals, thus enhancing the detection
of Erlotinib. Also, the FTMS’ superior mass accuracy
allows of better than 800 ppb allow for correct molecular
formula interpretation of MALDI Imaging results with high
confidence.
Conclusions
Mouse tissue samples containing the dosed cancer drug
Erlotinib were measured in vitro by mass spectrometry
imaging using both MALDI-TOF/TOF MS and MALDIFTMS. Erlotinib was detected in both the liver and kidney
tissue sections with each technique. MALDI-FTMS
generated MS data with high mass accuracy (better than
800 ppb) and high resolution (Figure 8). This technology
can be reliably used to proceed in more cancer or medical
research.
MALDI-FTMS delivers excellent mass
even under MALDI imaging conditions
Figaccuracy,
8
Fig. 8: MALDI-FTMS Mass
Accuracy and Isotopic
Distribution.
Stefen Laukien, The Rivers School (Weston, MA)
Co-Authors: Emily Creedon (The Rivers School), Jane-Marie Kowalski,
Paul Kowalski, Katherine Kellersberger (Bruker Daltonics),
Professor Nathalie Agar (Harvard Medical School).
Science Internship at Bruker Daltonics, Summer 2008
Keywords
Instrumentation & Software
Drug Imaging
ImagePrep
Tissue
autoflex III TOF/TOF
High Resolution Imaging
ultraflex III TOF/TOF
Drug distribution
apex-ultra FTMS
flexImaging
References
For research use only. Not for use in diagnostic procedures.
[1] Robert L. Comis. The Current Situation: Erlotinib
(ErlotinibErlotinib®) and Gefitinib (Iressa®) in Non-Small Cell Lung Cancer. The Oncologist, Vol. 10, No. 7, 467-470, August 2005.
[2] RX List, The Internet Drug Index,
http://www.rxlist.com/cgi/generic/ErlotinibErlotinib_cp-page3.htm
[3] Raj S. Pruthi, MD. Erlotinib Before and After Surgery in Treating Patients with Muscle Invasive Bladder Cancer. National Cancer Institute website: http://clinicaltrials.gov/ct2/show/NCT00380029 (updated Aug. 2008)
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The top trace shows a spectrum
of the measured mass of
Erlotinib, extracted from the
MALDI-FTMS liver tissue image.
The bottom trace shows the
simulated mass and isotopic
pattern of the molecular ion of
Erlotinib. This demonstrates
the excellent mass accuracy of
FTMS of better than 800 ppb
even under MALDI Imaging
conditions.