Presentation on the basic Maldi-Imaging workflow with some information on how it works.
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The document outlines the workflow and methodologies for MALDI-imaging, detailing the processes of tissue preparation, matrix selection, imaging techniques, and data analysis. It highlights the benefits, such as spatial profiling and the ability to analyze multiple compounds, as well as the drawbacks, including limitations in detecting larger proteins and accuracy in mass measurements. Various studies and optimization strategies from the literature are referenced to support the findings and techniques discussed.
![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](https://image.slidesharecdn.com/20140708presentationmaldi-imaging-140709051228-phpapp02/85/Presentation-on-the-basic-Maldi-Imaging-workflow-with-some-information-on-how-it-works-22-320.jpg)
![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](https://image.slidesharecdn.com/20140708presentationmaldi-imaging-140709051228-phpapp02/85/Presentation-on-the-basic-Maldi-Imaging-workflow-with-some-information-on-how-it-works-35-320.jpg)
![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](https://image.slidesharecdn.com/20140708presentationmaldi-imaging-140709051228-phpapp02/85/Presentation-on-the-basic-Maldi-Imaging-workflow-with-some-information-on-how-it-works-41-320.jpg)
Presentation on the basic Maldi-Imaging workflow with some information on how it works.
- 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
- 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.
- 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).
- 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
Editor's Notes
- #5: Tissue is snap frozen in liquid N2 Sections are cut using a cryostat and thaw mounted onto ITO-coated slides.
- #6: Sections are cut using a cryostat and thaw mounted onto ITO-coated slides.
- #7: Sections are cut using a cryostat and thaw mounted onto ITO-coated slides.
- #14: Sinapinic acid matrix (saturated in 50 : 50 acetonitrile/water) with increasing concentrations of TFA was deposited onto mouse liver tissue sections
- #23: Εκρόφηση, αποκόλληση Matrix-assisted Laser Desorption/Ionization Mass Spectrometry in Peptide and Protein Analysis J. Kathleen Lewis, Jing Wei, and Gary Siuzdak in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 5880–5894 Ó John Wiley & Sons Ltd, Chichester, 2000
- #24: Εκρόφηση, αποκόλληση Matrix-assisted Laser Desorption/Ionization Mass Spectrometry in Peptide and Protein Analysis J. Kathleen Lewis, Jing Wei, and Gary Siuzdak in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 5880–5894 Ó John Wiley & Sons Ltd, Chichester, 2000
- #25: Matrix-assisted Laser Desorption/Ionization Mass Spectrometry in Peptide and Protein Analysis J. Kathleen Lewis, Jing Wei, and Gary Siuzdak in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 5880–5894 Ó John Wiley & Sons Ltd, Chichester, 2000
- #26: Matrix-assisted Laser Desorption/Ionization Mass Spectrometry in Peptide and Protein Analysis J. Kathleen Lewis, Jing Wei, and Gary Siuzdak in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 5880–5894 Ó John Wiley & Sons Ltd, Chichester, 2000
- #29: After linezolid treatment, we found substantial overlap between the sites of kidney abscess formation as determined by MRI, and the abundance of calprotectin as determined by MALDI IMS ( Figure 5B and Movie S1 available online). In fact, calprotectin was only detectable within kidney abscesses, and not in the surrounding healthy tissue. In the untreated infected animals, kidney morphology was severely deformed and abscess architecture was difficult to discern. However, significant calprotectin signal could be detected through the infected organ at this time point suggesting the presence of a robust inflammatory response ( Figure 5D and Movie S2). Consistent with the reduction of inflammation imparted by linezolid, more calprotectin was detected in the kidneys of untreated animals as compared to treated animals. Additionally, the signal associated with truncated alpha-globin (m/z 5,020) was confined to the cortex and not detectable within the abscess in either the linezolid-treated or untreated mice ( Figures 5A and 5C and Movies S3 and S4).