1 matrix assisted laser desorption/ionization-mass spectrometry imaging (maldi-msi) for direct...
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Matrix assisted laser desorption/ionization-mass
spectrometry imaging (MALDI-MSI) for direct visualization of plant
metabolites in situ
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Presented by: Muhammadi Ph.D Scholar
Department of Botany, PMAS-UAAR, RWP.
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IntroductionMetabolomics has largely addressed through the
development of sophisticated separation techniques, mass spectrometry approaches, and computational tools
Little or no data regarding the original spatial distribution of metabolites in situ
Caprioli group pioneered MALDI-MSI-to visualize molecules directly in plant tissues and surfaces for the localization of lipids, proteins, secondary metabolites, and various small molecules at unprecedented spatial and chemical resolution.
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Improve spatial resolution and chemical coverage
Streamlined matrix and sample preparation
Easily accessible open-source image processing free-
ware
Other MSI platforms include:
a. Desorption electrospray ionization (DESI-MS)
b. Laser ablation electrospray ionization (LAESI-MS)
c. Secondary ion mass spectrometry (SIMS)
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Procedural overview Plant tissues are flash-frozen in an embedding media,
often gelatin
Cryo-sectioned and lyophilized
A chemical matrix, to promote desorption and ionization,
is applied by either a sprayer/nebulizer or by solvent-free
sublimation
Sample plate is inserted into the instrument
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Mass spectrum is produced
The resulting spectra at each location are used to
reconstruct MS images for ions of interest by
converting the ion’s intensity at every coordinate into
a color scheme.
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Localization of molecules in plant tissue sections and on tissue surfacesLipidsPhospholipids, comprising the lipid-bilayer of cell
membranes, and triacylglycerols (TAGs), 30% mass ∼of oil seeds, have been visualized by MSI
MALDI-MSI lipidomics studies in plant examined the spatial distributions and composition of the major and minor storage and membrane lipids (e.g., TAGs, phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and phosphatidic acid (PA) in embryos of upland Gossypium hirsutum
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Secondary metabolitesHortatines in mature barley seeds
Flavonols and dihydrochalcones in Golden Delicious
apple fruit sections
Lignan and cyanogenic glucoside-related metabolites,
coveted for their antioxidant activity, were
investigated by MALDI-MSI in developing Linum
usitatissimum
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In insect herbivore–plant interaction study, the metabolism of ingested glucosinolate, sinalbin, from Sinapsis alba (white mustard) leaves by Athalia rosae (turnip sawfly) was monitored using MALDI-MSI. MS images of longitudinal cryo-sections of these larvae revealed the rapid sequestration and concentration of sinalbin in the haemolymph, rather than gut, as a strategy to detoxify ingested leaf material
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Small molecule metabolitesMolecular weight <500 DaA challenge due to: Ion suppression by intense matrix peaks Susceptibility to in-source fragmentationPlant hormones (e.g., abscisic acid, indole acetic acid,
jasmonate, gibberellic acid etc)
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Metabolic incorporation of stable isotope labels
MALDI-MSI and SIMS - recycling of nitrogen by living plants from N15 enriched dead plant materials into N15choline and phosphocholine
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On plant surfaces
Unique capabilities of MALDI-MSI is that ions can
be desorbed/ionized directly off tissue surfaces.
Laser desorption ionization and colloidal silver used
to analyze the epicuticular lipids on plant surface
Laser beam cannot penetrate into cutin layers
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Technological advances Modification of laser optics : laser spot sizes <10 µm Map chemical heterogeneity by tissue type, cell to
cell, or even organelle to organelle basis.The Spengler group - range of 3 µm using a close-up
laser focusing in atmospheric pressure MALDI Caprioli group - 5 µm using modified laser optics Lee group demonstrated cellular/subcellular level
resolution MSI for juvenile Zea mays leaf cross sections at 5 µm spatial resolution
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MALDI-2Secondary step, ionizes molecules commonly lost during
the first laser desorption/ionization event
Increase the sensitivity and detection of low abundance
metabolites
Enable further reductions in spatial resolution, since less
energy accompanying smaller laser spot sizes will still
yield higher amounts of metabolites from the MALDI-2
event
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Serious bottlenecks in MSI imaging:
Low throughput
Lack of streamlined workflow for data analysis
Bruker commercialized MALDI-TOF MS
Fifty times faster than traditional mass spectro- meters
50 true pixels per second, using a 10 kHz laser
Scanning laser mirrors
Synchronized plate movement
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Efforts in computational tools may accelerate data
processing
Software is automatically perform statistical analysis
correlating the m/z ions that have similar image
patterns
www.scils.de
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Future perspectives
Development of new matrices and sample preparation, is
expected to evolve and improve in the coming years
Quantification in MSI is still a major hurdle
What is the physiological, biochemical, or developmental
significance of heterogeneity of tissue metabolites?
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Can this be further addressed with complementary
techniques such as in situ hybridization of mRNA or
MALDI-MSI of metabolic pathway enzymes?
What ways can MALDI-MSI be used to trace metabolism
over time?
Can quantification be routinely achieved with MALDI-
MSI?
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THANKS
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ReferenceSturtevant, D., Y. Lee, K. Chapman. 2015. Matrix
assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for direct visualization of plant metabolites in situ. Current opinion in Biotechnology 2016, 37:53–60
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