maldi ms imaging on the 4800 maldi tof/tof™ analyzer prepared by andrew james, phd
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MALDI MS Imaging on the 4800 MALDI TOF/TOF™ Analyzer
Prepared by Andrew James, PhD
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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.
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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
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Image Acquisition
• 4800 data• MSI imaging acquisition tool and 4000
Series Explorer used togetherSetup• Laser: 50 shots/pixel• Pixel size 200um x 200 um• Linear TOF acquisition method: 3-25 kDa
(note: No active processing method)
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4800 MALDI TOF/TOF
Slice Tissue SectionsMount Sample
Spray Coatwith Matrix
Load Sample
Acquisition Software
Data Processing
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Image Analysis
• Image analysis was carried out on BioMap software– Produce Images– Data manipulation (baseline correction on
underlying spectra)– Compare results images
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Active Image file Optical Image
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Active ImageWindow
MS Image result windows
Optical Image
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• 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
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Relative Intensity
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Image Overlay
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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
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Comparing Spectra in Different ROIs
• Draw ROI in the Active Image data window
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Define ROIs
Compare average spectra in different regions
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Compare Spectra from multiple ROIs
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• Useful for visualizing differences
• Can drag cursor over the over-layed spectra to generate images in the active image data window
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Comparing Intensity of an Analyte of Interest
• Draw ROIs within an MS image result window
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Compare intensity of detection protein in specific regions of sample
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Compare average intensity of a detected protein in specific regions of the sample
Average intensity of detected protein within thespecified ROI
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• Get statistics for average intensity of a specific mass in different regions of the tissue section– Relative quantitation
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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