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LIPID MAPS Lipidomics WorkshopApril 19, 2009
Future Directions:Tissue and Cell Imaging
Robert C. Murphy
Department of PharmacologyUniversity of Colorado Denver
www.lipidmaps.org
Other LIPID MAPS Bridge C (Imaging Group):University of Colorado Penn StateDenverRobert Barkley Nicolas WinogradJoseph Hankin Melissa Passarelli
m/z 806(16:0a/22:6-PC) [M+H]+
16:0/22:6 PCm/z 806.4
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Outline:A. Brief introduction of tissue molecular imaging by mass
spectrometryB. Sample preparation issues: tissue preparation, matrix applicationC. Compound identification: Molecular weight (high resolution) and
MS/MSD. Brain images-Abundance of Ions
i. Abundance of ions/abundance of phospholipidsii. Microdissection and LC/MS/MS quantitation
E. Buckyball (C60+) images
F. FutureG. References
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Maldi Plate
hυ337 nmN2-laser
Ions
Matrix: 2,5-dihydroxybenzoic acid
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MALDI- QStar (Q-TOF)
MALDI Imaging of Lipids
(Richard Caprioli-protein/peptides)
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Dry ice acetone-20o
Sublimation
2,5-DihydroxyBenzoic acid(matrix)
Tissue(Dried)
Vacuum
Sublimation
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Sample preparation
• Mouse brain flash frozen-70oC• Warmed to -15oC, mounted with Optimal cutting
temperature compound• Sliced (cyrostat) 10 um thickness• Placed directly on glass cover slips or MALDI
steel plate• Stored -20oC
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16:0a/18:1-PCm/z 760.6Positive ions
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m/z 760.6
m/z 798.6
m/z 826.6
m/z 734.6
m/z 651.5
m/z 577.5
Full Scan Spectrum of image
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Cerebral Cortex
Corpus Callosum
Thalamus
CerebellumHippocampus
Medulla
Hypothalamus
Pons
Striatum
m/z 760.6
100 300 500 700 900m/z
184.1
760.6
100
MS/MS
Rela
tive
Inte
nsity
Identification of Lipids (MS/MS)
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18:0a/22:6-PCm/z 834.6
Docosahexaenoic Acid Containing Phosphatidylcholine
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18:0a/20:4-PIm/z 885.6
d18:0/24:1 – STm/z 888.7
Stain imageAllen Brain Atlaswww.brainatlas.org
Negative ions
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18:0a/22:6-PSm/z 834.6
100 300 500 700 900m/z
Rela
tive
Int
ensi
ty
419.2
283.2
747.5437.2
152.9463.2 834.6327.2
100
[M-H]-
[M-H-87]-
MS/MS(CID)m/z 834.6
Negative ions
CerebellumA
B
Negative Ions
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What do the ion abundances mean?
• Phospholipid species is present?• Concentration is higher than other
regions?
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Is there more 16:0/16:0-PC ( m/z 734.4)in cortex than in corpus callosum?
16:0/16:0-PC 16:0/16:0-PC
CortexCortex
Corpuscallosum
Corpuscallosum
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16:0/22:6 PCm/z 806.4
Is there really more esterified 22:6 in the rat cerebellar grey matter?
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18:0/18:1 PC [M+H]+ 18:0/18:1 “DAG-like”+
Is there really more esterified 18:0/18:1 in the rat white matter?
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Do observed ions reflectactual concentrations?
• Image of major phospholipids from a slice• Isolate mouse brain regions (micro dissection)
on adjacent slice • Extraction (added 100ng deuterated-PC internal standard)
– Normal phase LC/MS (electrospray ionization)• Positive ions
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Abundance of ions in images:False negatives/no false positives
• Imaging of Phospholipids in tissue slices– Striking distribution of specific molecular species in
regions (50 micron resolution)– Observance of m/z means PL is present at site
• False negative information likely– Mechanism of lipid secondary ion release?– Abundance of ions
• Im/z = f([Lipid] x [ionization cross section] x [local environment] x …)
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Why is local environmentso different about white matter?
Myelin sheath
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369 527
561
651 734
760 798
788
806
826 R1R2
Total MALDI ions in celebellar grey(blue)and celebellar white (red)
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Collaboration :Nick Winograd Penn State
• SIMS (secondary ion mass spectrometry) based imaging– Buckyball ion beam (C60
+)– Better lateral resolution
• Lipid bilayers with SIMS (Ga+) Science, 2004• 200 nm ion beam
• Compare sublimation MALDI with SIMS– Prepare rat cerebellum on In oxide glass slides– Serial sections analyzed UCHSC/Penn State
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Buckyballs [C60]+Nick Winograd-Penn State
Weibel, Wong, Lockyer, Blenkinsopp, Hill and Vickerman, Anal. Chem., 2005.
Melissa Melissa PassarelliPassarelli
Primary ion beam focused to a submicron spot
Each carbon atomcarries 1/60th of totalincident kinetic energy
Other ion beams-Au3, Bi3, SF9, Au 400
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CC6060 Ion SourceIon Source
Spatial Resolution < 300 nmEnergy range 10 keV – 40 keVSource lifetime > 600 hours
IonoptikaIonoptika
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100 um
50 um
10 um
Cholesterol m.z 369PC head group m/z 184
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Conclusions
• Imaging of Lipids in the brain– Striking distribution of specific PL-molecular species
and even cationized species– Abundance information relevant within tissues of
similar cellular structure– Rich biochemical information– Can we improve lateral resolution?
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G. Examples of imaging lipids in tissues
Sublimation as a method of matrix application for mass spectrometric imaging. (2007). Hankin JA, Barkley RM, Murphy RC. J Am Soc Mass Spectrom 18:1646–1652
Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease (2008) Chen Y, Allegood J, Liu Y, Wang E, Cachón-Gonzalez B, Cox TM, Merrill AH Jr, Sullards MC. Anal Chem. 80(8):2780-8.
Molecular imaging of proteins in tissues by mass spectrometry (2008). Seeley EH, Caprioli RM Proc Natl Acad Sci;105:18126-31.
Nanoparticle-assisted laser desorption/ionization based mass imaging with cellular resolution.( 2008 )Taira S, Sugiura Y, Moritake S, Shimma S, Ichiyanagi Y, SetouM. Anal Chem. 80:4761-6.
Solvent-free matrix dry-coating for MALDI imaging of phospholipids.( 2008 ) PuolitaivalSM, Burnum KE, Cornett DS, Caprioli RM. J Am Soc Mass Spectrom. 19:882-6.
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Acknowledgements
UCDenver• Joseph Hankin• Robert Barkley• Santiago Farias
Penn State• Nick Winograd• Melissa Passarelli
MS-Imaging
National Institutes of HealthLipidMaps GM 044567
m/z 806(16:0a/22:6-PC) [M+H]+