Presentation on the basic Maldi-Imaging workflow with some information on how it works. This presentation was prepared for a group meeting and is focused almost entirely on the process of MALDI-Imaging to give the group leaders an understanding of the process as well as some important information on how to make it work well.

1. Diane Hatziioanou (Άρτεμις)
Postdoctoral Researcher

2. • MALDI-Imaging Workflow
• How MALDI-Imaging works
• MALDI-Imaging data information
• Preliminary results
• Comments on 2D protein electrophoresis

3. MALDI-Imaging Workflow

4. Workflow
Tissue preparation
•Tissue is snap frozen in liquid N2
Image from Wikipedia

5. Workflow
Tissue preparation
•Embedding material such as OCT is avoided as these polymers
suppress ion signals and create background signals in MALDI-MS
Embedded in OCT
No OCT
Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

6. Workflow
Slide preparation
•Tissue is cut using a cryostat into 5-15μm sections
•Tissue sections are thaw mounted onto ITO-coated slides

7. Workflow
Slide treatment
• Slides are:
• Desiccated to remove moisture
• Washed in Ethanol to remove salts and
contaminants
• Optionally washed in organic solvents to
remove lipids
• Scanned/imaged
• Coated with a matrix
Rat
Kidney

8. Workflow
Slide Imaging/Scanning
• Slides are:
• Scanned using a conventional scanner
• Photographed from a microscope(and patched
together)
• Scanned using an aperiscope
Rat
Kidney

9. Workflow
Matrix selection
• SA routinely used for higher
molecular weight proteins
• SA yields the best combination
of crystal coverage and signal
quality.
• CHCA used for lower MW peptide
species
• DHB used for both mass ranges in a
single experiment

10. Workflow
Matrix concentration selection
Effect of matrix
concentration on
crystallization and the
resulting mass spectra.
Solutions of SA in 50 : 50
acetonitrile/0.1% TFA
(A) 10 mg/ml
(B) 20 mg/ml
(C) saturated
(>30 mg/ml)
Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

11. Workflow
Matrix solution selection
• Water and organic solvent mixture allows both hydrophobic and water-soluble
(hydrophilic) molecules to dissolve into the solution
• Acetone
• Methanol
• Isopropanol
• Acetonitrile
• Ethanol
• The liquids vaporize, leaving co-crystallized matrix with analyte molecules.
• Co-crystallization is a key issue in selecting a proper matrix to obtain a good
quality mass spectrum of the analyte of interest
• Sample acidification with up to 0.2% TFA may improve spectra
• Detergents (eg 0.05% v/v Triton X-100 or SDS) may improve detection of membrane proteins,
hydrophobic proteins and also increase overall protein signal intensities
Mainini V, Angel PM, Magni F, Caprioli RM. Rapid Commun Mass Spectrom. 2011 Jan 15;25(1):199-204. doi:
10.1002/rcm.4850.

12. Workflow
Matrix solution selection
Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

13. Workflow
Matrix solution selection –TFA concentration
Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

14. Workflow
Detergent effect on Matrix
Mainini V, Angel PM, Magni F, Caprioli RM. Rapid Commun Mass Spectrom. 2011 Jan 15;25(1):199-204. doi:
10.1002/rcm.4850.

15. Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney
sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016
Workflow
Solvent/TFA effect on Matrix

16. Workflow
Solvent/TFA effect on Matrix
Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney
sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016
AcN
EtOH
MeOH

17. Workflow
Matrix solution Application Method
• Spotting
• Manual deposition
• CHIP-1000 spotting device (piezoelectric technology)
-200 μm
• Coating (delivery of a homogeneous layer of matrix over the entire tissue)
• Imageprep (vibrational vaporization) -50 μm
• SunCollect sprayer(pneumatic sprayer) -50 μm
• Thin layer chromatography (TLC) sprayer
• Two-step approaches:
• Sublimation followed by recrystallization 1-2 μm

18. Workflow
MALDI-Imaging using a Bruker autoflex speed
• Slides are inserted into the Imager
• Software settings are selected and a
scanned slide image is calibrated to
match the orientation of the
imaged slide.
• Protein standards, parts of the test
tissue and surrounding matrix are
hit with the laser to test its
performance.

19. Live video of slide
Grid for positioning laser/video
Laser results (Calibration protein)
Software settings

20. Workflow
MALDI-Imaging using a Bruker autoflex speed –part 2
• An area of tissue is selected for
Imaging
• Several areas can be selected from
up to 2 slides
• Imaging of the selected areas is
performed (1h –o/n)
• Imaging data can be viewed as
average spectra, average spectra of
sub-areas or distribution of single
mass peaks.

21. How MALDI-Imaging works

22. MALDI is a two-step process
– A UV laser beam triggers desorption.
• Matrix material absorbs UV laser light and the upper
layer (~1 μm) of the matrix material is ablated
– The ablated plume contains many species: neutral and ionized
matrix molecules, protonated and deprotonated matrix
molecules, matrix clusters and nanodroplets.
– Analyte molecules are ionized (protonated or
deprotonated) in the hot plume
– eg. [M+H]+ (added proton), [M+Na]+ (added sodium ion), [M-
H]- (removed proton)
Laser
Ionized analytes
Laser
Ionized analytes
J. Kathleen Lewis, Jing Wei, Gary Siuzdak, Peptides and Proteins 2006 DOI: 10.1002/9780470027318.a1621

23. Laser
Ionized analytes
Hillenkamp, Franz, and Jasna Peter-Katalinic, eds. MALDI MS. John Wiley & Sons, 2007.
High-speed time-lapse
photographs
of IR-MALDI plumes with
100-ns pulse width Matrix:
glycerol; time
resolution 8 ns; spatial
resolution 4μm.

24. Separation and detection of MALDI ionized analytes
(Mass spectrometry)
Ions’ Time Of Flight (TOF) analysis
• Ions are accelerated to a detector
• The arrival time at the detector is dependent
upon the mass, charge, and kinetic energy
(KE) of the ion.
– KE is equal to ½ mv2 (where v=velocity). Ions will
travel a given distance, d, within a time, t, where t is
dependent upon their mass-to-charge ratio (m/z)
– Increased resolution often comes at the expense of
sensitivity and a relatively low mass range(< 10 000
m /z)
Mass spectrometry -TOF Analyser
• Reflectors increase the mount of time (t) ions need to reach the detector while reducing
their KE distribution, thereby reducing the temporal distribution Δt.
• Resolution is defined by “peak mass” divided by “peak width” m /Δm (or t/ΔT). Increasing
t and decreasing Δt results in higher resolution.
• Once the mass spectrum is acquired the sample is moved by a defines distance and the
next position in the sample is analyzed the same way.
J. Kathleen Lewis, Jing Wei, Gary Siuzdak, Peptides and Proteins 2006 DOI: 10.1002/9780470027318.a1621

25. MALDI analyzers
• MALDI MS is
– most commonly combined with TOF mass analyzers.
– MALDI MS can alternatively be combined with Ultrahigh-resolution ( > 105) mass
analyzers such as the Fourier transform ion cyclotron resonance (ICR) mass
analyzer – called Fourier transform mass spectrometry (FTMS) .
Analyzer
Meyers, Robert A., ed. "Encyclopedia
of analytical chemistry." (2000).

26. MALDI-Imaging data information

27. MALDI-Imaging data information
MALDI-Imaging data can give spatial distribution patterns even at 200 μm
resolution
Bruker Daltonics Application Note # MT-91
Whole-organ MALDI Imaging

28. Whole-Animal MALDI Imagin
UninfectedInfected
Ahmed S. Attia,, et . al. Monitoring the Inflammatory Response to Infection through the
Integration of MALDI IMS and MRI, Cell Host & Microbe, 2012 (11) 664-73
H&E-stained sections of entire mice
Masses corresponding to proteins that are
abundant in the liver (m/z 3,562), kidney
(m/z 5,020), brain (m/z 10,258), or
systemically (m/z 11,837) in both infected
and uninfected mice are shown.
In addition, masses corresponding to
proteins that are only expressed in infected
animals are shown (m/z 10,165, 10,202,
10,369).

29. Separation and detection in MALDI-Imaging
• The 3D structure of the samples affects ion flight times and
results in significantly lower mass resolution and mass accuracy
• Mass deviations up to 0.5 m/z not uncommon.
• Spatial resolution typically 50-200 μm per pixel.
– Resolution up to 1 μm possible.
• Samples can be trypsin digested to detect larger molecules
– Matches with LC-ESI-MS/MS only possible with low ppm range mass
accuracy for both measurement models, less accurate measurements
lead to ambiguous assignments.
Römpp A, Spengler B. Histochem Cell Biol. 2013 Jun;139(6):759-83. doi: 10.1007/s00418-013-1097-6.

30. MALDI-Imaging Drawbacks
• Only detects the most abundant molecules
• Difficult to detect proteins over 20 kDa
• Identification of masses possible only with low
ppm range mass accuracy less accurate
measurements lead to ambiguous
assignments.
– But... Results and profile publishable without
identification.

31. MALDI-Imaging Benefits
• Spatial profiling
• Analysis of all parts of sample in one reading
• Untargeted (label free), multiplex method.
– Add desorbed and ionized compounds in the sample
are detected, regardless whether
known/unknown/expected /unexpected
• Can optimize conditions to detect proteins,
peptides, lipids, drug compounds a.o.
• Allows for investigation of disease formation,
progression, and treatment

32. Preliminary work

33. MALDI-Imaging work
• 2 MALDI-Imaging slides run
– Cryosectioning training obtained
– Instrument time and supervision not always
available
• Conditions used were those used in lab for
brain tissue.
– Imageprep used for matrix deposition
– Insufficient amount of matrix used.

34. MALDI-Imaging workNo PBS1
PBS
wash
(Background)
+35mg/ml SA
in 50:50 AcN
10mg/ml SA in 60:40 AcN,
0.2% TFA

35. MALDI-Imaging work
-Images from kidney taken from a dead rat.
30mg/ml SA in 70:30 AcN,
0.1% TFA
0
20
40
60
80
100
Intens.[a.u.]
5000 10000 15000 20000 25000
m/z
Control
0
20
40
60
80
Intens.[a.u.]
5000 10000 15000 20000 25000
m/z
Acetone washed
0
50
100
150
200
Intens.[a.u.]
5000 10000 15000 20000 25000
m/z
Chloroform washed

36. Comments on 2D protein
electrophoresis

37. 2D protein electrophoresis work
• Optimized conditions work very well
• Results (24 samples -without data analysis)
deliverable within 1-2 months

38. 2D protein electrophoresis work
12% SDS-PAGE
15--
kDa
180-
kDa
10% SDS-PAGE
15--
kDa
180-
kDa
11% SDS-PAGE
15--
kDa
180-
kDa

39. National and Kapodistrian University
of Athens
• Vlahakos Dimitrios
BRFAA
• Charonis & lab
– George Barkas
• Vlahou & lab
– Manousos Klados
– Makis Zoidakis
– Vasiliki Bitsika
Demokritos
• Tsilibary & lab
– Aspasia Volakaki
National and Kapodistrian
University of Athens
Università degli Studi di Milano-
Bicocca
• Magni & lab
– Andrew Smith
Many thanks to:

40. Sublimation Device
Joseph A. Hankin, Robert M. Barkley, and Robert C. Murphy J Am Soc Mass Spectrom. Sep 2007; 18(9): 1646–1652.

41. UV MALDI Matrix List
Compound Other Names Solvent
Wavelength
(nm)
Applications
2,5-dihydroxy benzoic
acid[1] DHB, Gentisic
acid
acetonitrile, water,
methanol, acetone,
chloroform 337, 355, 266
peptides, nucleotides,
oligonucleotides,
oligosaccharides
3,5-dimethoxy-4-
hydroxycinnamic
acid[2][3]
sinapic acid;
sinapinic acid;
SA
acetonitrile, water,
acetone,
chloroform 337, 355, 266
peptides, proteins,
lipids
4-hydroxy-3-
methoxycinnamic
acid[2][3] ferulic acid
acetonitrile, water,
propanol 337, 355, 266 proteins
α-Cyano-4-
hydroxycinnamic
acid[4] CHCA
acetonitrile, water,
ethanol, acetone 337, 355
peptides, lipids,
nucleotides
Picolinic acid[5]
PA Ethanol 266 oligonucleotides
3-hydroxy picolinic
acid[6] HPA Ethanol 337, 355 oligonucleotides

42. Workflow
Solvent/TFA effect on Matrix
Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney
sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016
AcN
EtOH
MeOH
MALDI images and spectra rat kidney sections (A) Male 3, (B)
Male 5, (C) Female 2, (D) Female 5 (E) intensity legend, where:
(i) image at m/z 15.3 with spectra from area highlighted within
tissue section,
(ii) spectrum from outside of the tissue boundaries and
(iii) image and spectrum at m/z 18.7.
i
ii
iii
i
ii
iii
i
ii
iii
i
ii
iii