Download 4800 MALDI TOF/TOF Slice Tissue Sections Mount Sample

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)
Slice Tissue Sections
Mount Sample
Data Processing
Spray Coat
with Matrix
4800 MALDI
TOF/TOF
Load Sample
Acquisition Software
Image Analysis
• Image analysis was carried out on BioMap
software
– Produce Images
– Data manipulation (baseline correction on
underlying spectra)
– Compare results images
Active Image file
Optical 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
Compare average spectra in different regions
Define ROIs
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