Transcript of "Molecular analysis of whole body animal tissue secitions by imaging maldi ms"
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
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
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
Fragmentation of Olanzapine and MetabolitesConfidential 11
Glioma Tumor Mouse Section• Tumor specific protein signals were detected• Proteomic information was extractedConfidential 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
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
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
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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