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Tissue Imaging for the Masses

Tissue Imaging for the Masses. ASMS - June 4 2008. Concept of MALDI Tissue Imaging MS. 1. Section tissue sample. 3. Acquire data from whole sample. 4. Select peaks of interest and display intensities. 2. Deposit MALDI matrix. Sample preparation is the key.

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Tissue Imaging for the Masses

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  1. Tissue Imaging for the Masses ASMS - June 4 2008

  2. Concept of MALDI Tissue Imaging MS 1. Section tissue sample 3. Acquire data from whole sample 4. Select peaks of interest and display intensities 2. Deposit MALDI matrix

  3. Sample preparation is the key • Sample integrity is very important • Sample washing is necessary for removing unwanted compounds. • OCT - water wash (2x 80C Milli Q water 1min) • Lipids - chloroform (1x 30 seconds) • FFPE de-waxed - gently immersing in xylene for 10 minutes pre-warmed to specified temperature in water bath. • Washed in stepwise immersion, 5 minutes duration each: • 2x 100% ethanol • 1x 90% ethanol • 1x 80% ethanol • 1x 70% ethanol • Trypsin / matrix deposition method is critical to highly sensitive results. Aoki et al. (2007) Proc. Jpn. Acad., Ser. B83, 205-214

  4. 55µm Orifice 100 pl Droplet CHIP-1000 Chemical Printer A. Piezo electric device B. One shot vessel (OSV) C. Detachable vacuum and pressure manifold C B A Shima et al. (2006) Journal of Mass Spectrometry Society Japan, 54, 4, 133-140

  5. CHIP print head OSV volume up to 500ul “Dead volume” ~50-100ul

  6. Chemical printer - Operation • Printing Features: • Adjustable X, Y printing pitch • Can set positions µm apart or mm apart • Right: 50% 2-proponal jetted in 10x10 arrays • Centre-to-Centre distance decreased from 500µm to 120µm

  7. What resolution is really possible? • Decide on desired total volume for good results. • Example – 8nl per print position • Decide on resolution • Example 1000um centre to centre • Decide on number of drops per round • Example 20 drops (2nl per round) • (1 drop = 100pl) • Decide on waiting step for solvent drying FIXED to 100pl Drops set by user

  8. Iterative printing - benefits • Iterative printing enables higher volumes to be dispensed to a single print position. • Spatial resolution is maintained without print positions spreading.

  9. Typical experiments • Matrices • Target – protein (10-20nl/pp) • 5 mg/mL sinapinic acid, 50% ethanol, 0.1% TFA • Target - peptide (40nl/pp) • 50ug/ml trypsin, 25mM ammonium bicarbonate, followed by 3h incubation in total humidity at 37C. • 5 mg/ml CHCA, 75% methanol • Or • 8-20 mg/mL DHB, 50% ethanol, 0.1% TFA (can go up to 50mg/ml if necessary) • Spatial resolution to quantity of drops ratio • 10 drops – 600-700um Printing time short • 5 drops – 250-300um • 2 drops – 120-150um Printing time long Andersson et. al (2008) Nature Methods, Vol 2 No 1, 101-108

  10. CHCA 40nl per print position 5 drop per rounds (500pl) Repeated 80x Enables 250um resolution

  11. MALDI data acquisition • Take print location file from the CHIP

  12. Set up laser roaming parameters 200 shots per print position 20 shots per movement 10 positions 200um

  13. Laser Roaming 200 shots per print position 20 shots per movement 10 positions 200um

  14. Laser roaming – data quality improved • Standard protein mixture printed using CHIP-1000 • 10x10 print positions (pp) spaced at 250um, 20nl/pp printed 5 drop iterations. • Data acquisition: 200 laser shots/pp • With laser roaming – 60um pp radius, 10 shots per movement • Data acquired 3x Laser roaming gives highest intensity data With Roaming 1st acquisition With Roaming 2nd acquisition With Roaming 3rd acquisition No Roaming 1st acquisition No Roaming 2nd acquisition No Roaming 3rd acquisition

  15. With roaming Without roaming MALDI images (BioMap) • Relative ion intensities are more homogeneous over the scanned area. • No laser roaming leads to some data being very low in intensity. • Good quality data can still be acquired for the third data acquisition. With Roaming 1st acquisition With Roaming 3rd acquisition No Roaming 1st acquisition No Roaming 3rd acquisition

  16. MALDI imaging - sensitivity • 10nl/pp of 25fmol/ul BSA digest solution (Red) • 1nl/pp of 25fmol/ul BSA digest solution (White). • Peptides could clearly be observed in spectra and peaks could be selected to generated heat map. • This equates to 250 and 25 amol per print position! • Peptide – 1638 Da +/- 10 Da:

  17. MALDI Imaging – applications • Drug localisation in tissue samples • Endogenous protein analysis (Alzheimer's disease) • On-tissue digests, MSMS peptide analysis, database ID. • Biomarker discovery • Matrix crystallisation studies • Crime Scene Investigation – imaging human fingerprints • LC-MALDI – MS chromatogram

  18. Imaging Mouse spinal cord • Mouse spinal cord section • 20nl per print position printed at 250 um centre to centre (SA 10mg/ml in 50% acetonitrile, 0.1% TFA • Data Courtesy of Etienne Waelkens & Raf van der Plas – University on Leuven

  19. Mouse brain Neuropeptides • Low molecular weight neruropeptides ionise very well for MALDI imaging. • Research in brain related diseases could benefit from this extra information. 14000 Da 4955 Da 11350 Da 6710 Da CHIP scanned image Rohner et. al. (2005) Mechanisms of Ageing and Development, 126, 177-185

  20. MALDI MSI rat hearts - MS/MS analysis • Rats injected with Isoprenaline (induces heart attacks) • MALDI imaging MS analysis of digested heart sections • MSMS analysis identified Collagen Type I. H&E stained sample 936 Da ASMS 2008 poster – WPH-168

  21. Matrix crystallisation studies • MALDI matrices can crystallise in different ways • DHB large crystals • CHCA small crystals • MALDI imaging can be used to help analyse hot spots in sample spots and enable optimisation of solvent composition and concentrations Standard peptide mixture manually spotted using CHCA Spotted area

  22. Human fingerprint analysis • Fingerprint analysis has the potential to pick up small molecules such as drugs or lipids. • Matrix – CHCA, dusted over fingerprint. • Common MALDI peaks displayed in image here 172, 190, 212 and 379 m/z

  23. LC-MALDI - ion intensity mapping • Imaging of mass range 1000-3000 Da • Shows the eluted peptides

  24. LC-MALDI – distribution of a single mass • Select a single peptide and see the distribution / elution of a peptide

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