MALDI-IMS Tissue Analysis• Goal   – Gain knowledge of location of proteins and drugs in different cellular      regions of...
MALDI-TOFConfidential        3
Sample Preparation and MS Analysis• 10 week old male Fischer 344 rats   – Control and olanzapine, PO, 8 mg/kg   – Sacrific...
Other Experimental Notes• Cyt c, insulin β-chain, and apomyoglobin were used to  calibrate the MS• A standard curve of ola...
Sample Prep AndAnalysisConfidential   6
Confidential   7
Protein Analysis of Forebrain SectionConfidential          8
Protein Analysis of Other Tissue                          SectionsConfidential                  9
Whole Body Protein AnalysisConfidential                10
Fragmentation of Olanzapine and                  MetabolitesConfidential            11
Glioma Tumor Mouse Section• Tumor specific protein signals were detected• Proteomic information was extractedConfidential ...
OLZ and  Metabolites  in Rat (2 hr) Olanzapine Lung > spleen > bladder  N-desmethyl-OLZ (8%)  Liver > kidney > bladder2-hy...
OLZ and Metabolites in Rat (6 hr)    Olanzapine    66% less signal in    brain than 2hr  N-desmethyl-OLZ (13%)2-hydroxymet...
Summary• MALDI-IMS can be used to identify proteins, drugs, and  drug metabolites in various tissues and whole body       ...
Other Applications Since 2006• Improvements in methodology   – No need for flash freezing   – Preservation of tissue using...
Molecular analysis of whole body animal tissue secitions by imaging maldi ms
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Molecular analysis of whole body animal tissue secitions by imaging maldi ms

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Transcript of "Molecular analysis of whole body animal tissue secitions by imaging maldi ms"

  1. 1. 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
  2. 2. MALDI-TOFConfidential 3
  3. 3. 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 imagerConfidential 4
  4. 4. 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 platesConfidential 5
  5. 5. Sample Prep AndAnalysisConfidential 6
  6. 6. Confidential 7
  7. 7. Protein Analysis of Forebrain SectionConfidential 8
  8. 8. Protein Analysis of Other Tissue SectionsConfidential 9
  9. 9. Whole Body Protein AnalysisConfidential 10
  10. 10. Fragmentation of Olanzapine and MetabolitesConfidential 11
  11. 11. Glioma Tumor Mouse Section• Tumor specific protein signals were detected• Proteomic information was extractedConfidential 12
  12. 12. OLZ and Metabolites in Rat (2 hr) Olanzapine Lung > spleen > bladder N-desmethyl-OLZ (8%) Liver > kidney > bladder2-hydroxymethyl-OLZ (13%)Bladder > liver > kidney Confidential 13
  13. 13. 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
  14. 14. 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 levelsConfidential 15
  15. 15. 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 scanConfidential 16
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