MALDI MS Imaging on the 4800 MALDI TOF/TOF™ Analyzer Prepared by Andrew James, PhD

MALDI Imaging Mass Spectrometry (IMS) The analysis of a sample surface for its molecular content –Sample consists of a thinly sliced section of tissue/organ/whole animal A 2 dimensional array of MALDI spectra are obtained over the surface of the sample –each spectra has location component An ion intensity map can then be produced for any mass that is detected over the scanned area.

Sample Preparation Coronal Rat Brain Sections –12 um thickness After sample mounting, tissue sections were washed in 70% EtOH solution for 30 sec –Then dried under vacuum Tissue sections were spray coated with CHCA (10 mg/mL 60% Acn, 40%water, 0.1%TFA) –Approx.15 coats

Image Acquisition 4800 data MSI imaging acquisition tool and 4000 Series Explorer used together Setup Laser: 50 shots/pixel Pixel size 200um x 200 um Linear TOF acquisition method: 3-25 kDa (note: No active processing method)

4800 MALDI TOF/TOF Slice Tissue Sections Mount Sample Spray Coat with Matrix Load Sample Acquisition Software Data Processing

Image Analysis Image analysis was carried out on BioMap software –Produce Images –Data manipulation (baseline correction on underlying spectra) –Compare results images

Active Image fileOptical Image

Active Image Window MS Image result windows Optical Image

Active image window –Contains all the spectral information –Use to generate specific MS images MS image results windows –Intensity map image for a specific mass (protein or bio-molecule) Optical images –Scanned image of sample prior to matrix application

Relative Intensity

Image Overlay

Regions Of Interest (ROI) Compare Spectra from 2 different areas of tissue sample Compare intensities of a given compound in 2 different areas of tissue

Comparing Spectra in Different ROIs Draw ROI in the Active Image data window

Define ROIs Compare average spectra in different regions

Compare Spectra from multiple ROIs

Useful for visualizing differences Can drag cursor over the over-layed spectra to generate images in the active image data window

Comparing Intensity of an Analyte of Interest Draw ROIs within an MS image result window

Compare intensity of detection protein in specific regions of sample

Compare average intensity of a detected protein in specific regions of the sample Average intensity of detected protein within the specified ROI

Get statistics for average intensity of a specific mass in different regions of the tissue section –Relative quantitation

Conclusion Still some work to do with sample preparation –Should be seeing more complex spectra –Compare spotting vs. spraying of matrix –Surface charge affects? Data acquisition may be improved by using an rastering pattern instead of a stationary shot pattern over the sample Biomap is a very good processing tool