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

Confidential 3

MALDI-TOF

Confidential 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 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 6

SamplePrepAnd

Analysis

Confidential 7

Confidential 8

Protein Analysis of Forebrain Section

Confidential 9

Protein Analysis of Other Tissue Sections

Confidential 10

Whole Body Protein Analysis

Confidential 11

Fragmentation of Olanzapine and Metabolites

Confidential 12

Glioma Tumor Mouse Section• Tumor specific protein signals were detected

• Proteomic information was extracted

Confidential 13

OlanzapineLung > spleen > bladder

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

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

OLZ and Metabolites in Rat (2 hr)

Confidential 14

OLZ and Metabolitesin Rat (6 hr)

Olanzapine66% less signal in brain than 2hr

N-desmethyl-OLZ (13%)

2-hydroxymethyl-OLZ (15%)

Confidential 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 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