Transcript (MALDI-MSI) for direct visualization of plant metabolites in situ

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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Introduction
Metabolomics 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 surfaces
Lipids
 Phospholipids, 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 8
Secondary metabolites
 Hortatines 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 metabolites
Molecular weight <500 Da
A challenge due to:
 Ion suppression by intense matrix peaks
 Susceptibility to in-source fragmentation
Plant 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-2
 Secondary 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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Reference
 Sturtevant, 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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