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

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MALDI-TOF

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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

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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

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SamplePrepAnd

Analysis

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Protein Analysis of Forebrain Section

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Protein Analysis of Other Tissue Sections

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Whole Body Protein Analysis

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Fragmentation of Olanzapine and Metabolites

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Glioma Tumor Mouse Section• Tumor specific protein signals were detected

• Proteomic information was extracted

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OlanzapineLung > spleen > bladder

N-desmethyl-OLZ (8%)Liver > kidney > bladder

2-hydroxymethyl-OLZ (13%)Bladder > liver > kidney

OLZ and Metabolites in Rat (2 hr)

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OLZ and Metabolitesin Rat (6 hr)

Olanzapine66% less signal in brain than 2hr

N-desmethyl-OLZ (13%)

2-hydroxymethyl-OLZ (15%)

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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

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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