Small Molecule Drug Imaging of Mouse Tissue by MALDI-TOF/TOF Mass Spectrometry and FTMS

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1 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

2 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

3 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.

4 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.

5 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 MALDI- TOF/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 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 MALDI- FTMS (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 MALDI- FTMS. 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.

6 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , MT-93/FTMS-38 MALDI-FTMS delivers excellent mass accuracy, even under MALDI imaging conditions Fig. 8: MALDI-FTMS Mass Accuracy and Isotopic Distribution. 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. Authors 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 References [1] Robert L. Comis. The Current Situation: Erlotinib (ErlotinibErlotinib ) and Gefitinib (Iressa ) in Non-Small Cell Lung Cancer. The Oncologist, Vol. 10, No. 7, , August [2] RX List, The Internet Drug Index, [3] Raj S. Pruthi, MD. Erlotinib Before and After Surgery in Treating Patients with Muscle Invasive Bladder Cancer. National Cancer Institute website: (updated Aug. 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 For research use only. Not for use in diagnostic procedures. Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) Fax +1 (978) ms-sales@bdal.com Bruker Daltonik GmbH Bremen Germany Phone +49 (421) Fax +49 (421) sales@bdal.de