2. MALDI-IMS Tissue Analysis
• Goal
– Gain knowledge of location of proteins and drugs in different cellular
regions of tissue (e.g. olanzapine in rat brains)
• Tissue distribution is required for FDA
• Advantages over autoradiography
– Analysis of parent and metabolites (MW specific data)
– Can determine protein changes (100 kDa)
– No radioactivity required
• Amenable to HTS
– 1,000 spectra/s
– 100’s of proteins monitored in a single experiments
• Relatively simple and robust sample preparation
– No tissue homogenization required
Confidential 2

3. MALDI-TOF
Confidential 3

4. Sample Preparation and MS Analysis
• 10 week old male Fischer 344 rats
– Control and olanzapine, PO, 8 mg/kg
– Sacrificed at 2 and 6 hr post-dose
– Frozen in a block of hexane/dry ice and stored at -20C
• Frozen tissue is cut into sections 5-20 µm thick
• Thaw mounted on a MALDI plate
• Matrix is applied using an automated spotter (~ 100 pL droplets)
– 30-50 µm in diameter
– Average diameter of a mammalian cell is ~ 10 µM
• MS and MS/MS was acquired by MALDI-TOF
– x,y coordinates
• Density map of compounds and proteins is generated using an imager
Confidential 4

5. Other Experimental Notes
• Cyt c, insulin β-chain, and apomyoglobin were used to
calibrate the MS
• A standard curve of olanzapine was generated using 0.02-20
pmol/µL in the matrix
– MS and MS/MS data was collected
– Detection limit was 60 ng
• First tested liver, kidney, and brain tissues to develop the
protocol
– And identify protein signals unique for each tissue
• Whole body imaging was done using 4 MALDI plates
Confidential 5

6. Sample
Prep
And
Analysis
Confidential 6

7. Confidential 7

8. Protein Analysis of Forebrain Section
Confidential 8

9. Protein Analysis of Other Tissue
Sections
Confidential 9

10. Whole Body Protein Analysis
Confidential 10

11. Fragmentation of Olanzapine and
Metabolites
Confidential 11

12. Glioma Tumor Mouse Section
• Tumor specific protein signals were detected
• Proteomic information was extracted
Confidential 12

13. OLZ and
Metabolites
in Rat (2 hr)
Olanzapine
Lung > spleen > bladder
N-desmethyl-OLZ (8%)
Liver > kidney > bladder
2-hydroxymethyl-OLZ (13%)
Bladder > liver > kidney
Confidential 13

14. OLZ and
Metabolites
in Rat (6 hr)
Olanzapine
66% less signal in
brain than 2hr
N-desmethyl-OLZ (13%)
2-hydroxymethyl-OLZ (15%)
Confidential 14

15. Summary
• MALDI-IMS can be used to identify proteins, drugs, and
drug metabolites in various tissues and whole body
– First time this has been demonstrated
– Metabolites comprised ~20-30% of the MS/MS signal from
2-6 hr
• There was a high degree of consistency
– Low inter-animal variability, high reproducibility
• Detailed analysis of specific tissues is also possible
– Can monitor dynamic changes in protein levels
Confidential 15

16. Other Applications Since 2006
• Improvements in methodology
– No need for flash freezing
– Preservation of tissue using ethanol and paraffin
freezing
• Protein analyses by MALDI-IMS
– Normal vs. diseased tissue
– Protein degradation/modification
• MALDI-FTICR-MS of drugs and metabolites in tissues
– No need for MS/MS fragmentation and can scan
for hundreds of proteins in a single scan
Confidential 16