Mass spectrometry Technology in Proteomics...
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Mass spectrometry Technology in Proteomics MALDI Imaging Rabah Soliymani University Of Helsinki Protein Chemistry Unit Biomedicum – Helsinki [email protected]
Transcript of Mass spectrometry Technology in Proteomics...
Mass spectrometryTechnology in Proteomics
MALDI Imaging
Rabah Soliymani
University Of HelsinkiProtein Chemistry UnitBiomedicum – Helsinki
DefinitionDefinition
MS is an analysis technique to determine bymeans of a mass spectrometer the moleculemass of free ions in high vacuum
MALDI PrinciplesMALDI PrinciplesMix analytes with excess matrix compound to crystallize(1:1000 or 10 000)
Matrix absorbs at the same laser wave length(commercially available N2-laser@337nm, YAG laser@355nm, infrared [email protected])
Short waved laser pulses
Gas phase
Schematic diagram of a linear MALDI-TOF mass spectrometer.Delayed extraction (pulsed ion extraction) occurs at the ion source
Schematic diagram of a Reflector MALDI-TOF mass spectrometer.
MALDI-TOF = Matrix Assisted Laser Desorption/Ionization-Time Of Flight
N2 , 337nm
MALDIMALDI--TOF/TOFTOF/TOF forfor LIDLID--LIFT and highLIFT and high energyenergy CIDCID--LIFTLIFT
Target
LIFTPrecursorIon Selector
CID cell(e.g. Argon)
Potential Profile during LIFT
LIFT
1189
.557
1309
.633
1063
.618
842.
526 22
59.9
94
1149
.665
1261
.628
1442
.704
1527
.760
2030
.844
2211
.084
1966
.921
1021
.559
1098
.544
2385
.894
1826
.942
2298
.162
0
2000
4000
6000
8000In
tens
. [a.
u.]
1000 1500 2000 2500 3000m/z
Typical mass spectrum from a protein band trypsin digest
IsotopicIsotopic distributiondistribution of the peptide with monoisotopicmonoisotopic massmass ofof 1189.557 Da
1189
.557
1190
.556
1191
.564
1192
.572
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
1190 1192 1194 1196 m/z
1189.557 All C-atoms in the peptide molecule are C12
(monoisotopic mass)
1190.556 The peptide molecule contains one C13 atom
1191.564 The peptide molecule contains two C13 atoms
1192.572 The peptide molecule contains three C13 atoms
MALDIMALDI ImagingImaging (MALDI(MALDI--IMS)IMS)
Application: Tissue imaging
Definitions:MALDI Imaging (MALDI-IMS)
A technique for analyzing the spatial arrangement of proteins, peptides,lipids, and small molecules in biological tissues
A protein profiling technique which enables the direct sampling ofhistological section
A technology that utilizes MALDI MS to map molecules of interest in thintissue sections
Potentially can deliver highly parallel, multiplexed data on the specific localizationof molecular ions in tissue samples directly, and to measure and map the variationsof these ions during development and disease progression or treatment
Tissue section(mouse brain)
2,000 15,000 30,000
Ion
inte
nsity
Acquisition x
Acq
uisi
tion
ym/z
12mm
PrinciplesPrinciples
A laser is rastered over a defined area while acquiring a complete mass spectrumfrom each position, resulting in molecular images for multiple analytesCornett, et al., Nature Methods 2007
Adapted fromMinerva L., et al.,Proteomics 2008
IMS workflow
Stoeckli, M, et al., Analytical Biochemistry 2002
KeyKey stepssteps inin tissuetissue imagingimaging
Review: Goodwin, et al., Proteomics 2008
• Peptide digestion on the membrane (identificationof the proteins)• Direct trypsin spraying; (Jardin-Mathé et al., 2008)
Peptide IMS of pancreatic tissue ofnormal mouse
IMS image for ion m/z 3120 (insulin C peptide)
Islet-specific spectrum
Minerva, et al., Proteomics 2008
IMS vs.IMS vs. histochemicalhistochemical stainstain
IMS image for ion m/z 3120 (insulin C peptide) Overlay image of ion m/z 3120 and Methylene bluestained of the pancreas
Thiery, G., et al., Proteomics 2008
Imaging of regions immunoreactive with anti-synaptophysin Ab in healthy human pancreas.
(A) Localization of synaptophysin positive cells by TAMSIM. The monoclonal rabbit anti-synaptophysin is conjugated with the tag El 307 (498 m/z). The false color green points inthe section show the presence of the tag El 307 and thus synaptophysin positive cells.
(B) Classical IHC image with the anti-insulin Ab. The dark pink spots correspond toLangerhans islets and so the synaptophysin-positive cells. The distribution ofsynaptophysin positive cells in (A) is very similar to that in (B).
Comparing Regions of interestComparing Regions of interest
Control vs. patientControl vs. patient
Imaging MS for studying analyte deposition inImaging MS for studying analyte deposition indried droplet methodsdried droplet methods
•Distribution of analytes / particles indried droplet deposition is not even.
• Typical case: stronger deposition atedges (coffee droplet effect).
• Matrix-free ionization of peptides.
1 mm circle and square shaped spots
Raster spacing 25 µm or 50 µm
Some preliminary resultsSome preliminary results
Angiotensin 2, 1 µl, 1 pmol
1 mm spot [A+H]+
2 mm spot [A+H]+
2 mm spot [A+K]+
Some preliminary resultsSome preliminary results
Angiotensin 2, 1 µl, 1 pmol
1 mm spot [A+H]+
2 mm spot [A+H]+
Divider/concentrator spot
A whole mouse
Penetration of the drug into the tissue
LimitationsLimitations,, soso farfar ……
Scans are for <30 kDa rangesSample preparations challenges• Tissue cryosections (mostly)• Autolysis possible• Tissue fixation• Suppression effect
Interpretation of complex spectra is not straight forward• Protein identifications challenges• Possible displacement or loss of small molecules after trypsinization for
MSMSMinor proteins enrichment is a handicapRequires other staining methods for comparison
