presentation on the basic maldi-imaging workflow with some information on how it works
DESCRIPTION
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.TRANSCRIPT
Matrix-assisted LaserDesorption/Ionization
(MALDI) -ImagingDiane Hatziioanou (Άρτεμις)
Postdoctoral Researcher
• MALDI-Imaging Workflow • How MALDI-Imaging works• MALDI-Imaging data information• Preliminary results• Comments on 2D protein electrophoresis
MALDI-Imaging Workflow
Workflow Tissue preparation
•Tissue is snap frozen in liquid N2
Image from Wikipedia
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.
Workflow Slide preparation
•Tissue is cut using a cryostat into 5-15μm sections•Tissue sections are thaw mounted onto ITO-coated slides
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
RatKidney
Workflow Slide Imaging/Scanning
• Slides are:• Scanned using a conventional scanner• Photographed from a microscope (and patched
together)
• Scanned using an aperiscope
RatKidney
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
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.
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.
Workflow Matrix solution selection
Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.
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.
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.
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
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
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
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.
Live video of slide
Grid for positioning laser/video
Laser results (Calibration protein)
Software settings
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.
How MALDI-Imaging works
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
Laser
Ionized analytes
Hillenkamp, Franz, and Jasna Peter-Katalinic, eds. MALDI MS. John Wiley & Sons, 2007.
High-speed time-lapse photographsof IR-MALDI plumes with 100-ns pulse width Matrix: glycerol; timeresolution 8 ns; spatial resolution 4μm.
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
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).
MALDI-Imaging data information
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
Whole-Animal MALDI ImaginUninfectedInfected
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).
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.
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.
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
Preliminary work
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.
MALDI-Imaging workNo PBS1
PBS wash
(Background)
+35mg/ml SA in 50:50 AcN
10mg/ml SA in 60:40 AcN, 0.2% TFA
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
Inte
ns. [a
.u.]
5000 10000 15000 20000 25000m/z
Control
0
20
40
60
80
Inte
ns. [
a.u.
]
5000 10000 15000 20000 25000m/z
Acetone washed
0
50
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200
Inte
ns. [a
.u.]
5000 10000 15000 20000 25000m/z
Chloroform washed
Comments on 2D protein electrophoresis
2D protein electrophoresis work
• Optimized conditions work very well• Results (24 samples -without data analysis)
deliverable within 1-2 months
2D protein electrophoresis work
12% SDS-PAGE
15--kDa
180-kDa
10% SDS-PAGE15--kDa
180-kDa
11% SDS-PAGE
15--kDa
180-kDa
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:
Sublimation Device
Joseph A. Hankin, Robert M. Barkley, and Robert C. Murphy J Am Soc Mass Spectrom. Sep 2007; 18(9): 1646–1652.
UV MALDI Matrix ListCompound 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, 266peptides, proteins,
lipids4-hydroxy-3-
methoxycinnamic acid[2][3] ferulic acid
acetonitrile, water, propanol 337, 355, 266 proteins
α-Cyano-4-hydroxycinnamic acid[4]
CHCAacetonitrile, water, ethanol, acetone 337, 355
peptides, lipids, nucleotides
Picolinic acid[5] PA Ethanol 266 oligonucleotides3-hydroxy picolinic
acid[6] HPA Ethanol 337, 355 oligonucleotides
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.
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ii
iii
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ii
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