imaging mass spectrometry (ims): searching for the...
TRANSCRIPT
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Imaging Mass Spectrometry (IMS): Searching for the invisible,a new tool for disease investigations
Meilahti Clinical Proteomics Unit (MCPU)University of Helsinki, Faculty of Medicine
Biomedicum Helsinki and Haartman InstituteE-Mail: [email protected]
(http://research.med.helsinki.fi/corefacilities/proteinchem)
GPF/Göttingen 11/2012
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Mass Spectrometry Imaging (tissue, cells, bacteria,
viruses...)
Equals scanning with a mass spectrometer the invisible
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Definition
MS is an analysis technique to determine by means of a mass spectrometer the molecule mass of free ions in high (semi high) vacuum
i.e. it sees everything with a mass!
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MALDI Principles
• Mix 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
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MALDI-TOF/TOF for LID-LIFT and high energy CID-LIFT
Target
LIFT Precursor Ion Selector
CID cell(e.g. Argon)
Potential Profile during LIFT
LIFT
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MALDI TOF/TOF
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HR Quadrupole TOF/TOFWith MALDI and ETD
Ion Mobility
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A 384 position MALDI-TOF sample target plate. To each position 0.5-2µl of sample together with matrix solution is pipetted and allowed to dry.
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Tissue slides for IMS-MS
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Tissue slide for IMS-MS
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5-200 µm raster step
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PrinciplesTissue section (mouse brain)
2,000 15,000 30,000
Ion
inte
nsity
Acquisition x
Acq
uisi
tion
ym/z
12mm
• A laser is rastered over a defined area while acquiring a complete mass spectrum from each position, resulting in molecular images for multiple analytesCornett, et al., Nature Methods 2007
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Schematic representation of the MALDI-MSI work flow
Franck J et al. Mol Cell Proteomics 2009;8:2023-2033
©2009 by American Society for Biochemistry and Molecular Biology
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MSI workflow
Stoeckli, M, et al., Analytical Biochemistry 2005
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• Definitions:
• MALDI Imaging (MALDI-IMS)(MS-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 of histological section
• A technology that utilizes MALDI MS to map molecules of interest in thin tissue sections
• Potentially can deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment
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Benefits of MALDI-MSI
• Analysis of entire sample in one reading
• Previous knowledge of molecular composition is not necessary
• Allows for investigation of disease formation, progression, and treatment
www.maldi-msi.org
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MS imaging advantages
•No labeling requiredBiomolecules are functionally unmodified
•Image biomolecular modificationsPTM’s, Metabolites
•Detailed information on molecular identity
•Large scope of different elements and molecules
Targeted labeling
Label free imaging
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MSI vs. histochemical stain
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Imaging Endogenous peptides Imaging Gangliosides
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New Phytologist (2007) 173 : 438 –444m/z 381
m/z 543
m/z 705
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Heeren R, USA
Collection and storage of samples
Tissue preparation and matrixapplication
Section mounting
Fixed versus fresh tissue
Embedding
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Collecting the samples
1. Sample handling and preparation of sections for image analysis are critical to the spatial integrity of measured molecular distributions.
2. Any molecular degradation that occurs in the time between sample collection and analysis can adversely affect the results.
3. A typical study may involve samples collected over a lengthy period of time, and standardized procedures are therefore required to minimize experimental variability over the time course of the study.
4. Good communication among all personnel involved with collecting, storing and analyzing samples is critical.
5. Ideally, samples are frozen immediately after collection and stored at -80°C until sections for MALDI-IMS analysis are cut on a cryomicrotome just before analysis.
Cornett et al., Nature Methods, 2007
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Preserving the tissues1. Animals are usually killed by cervical dislocation, after which the tissue of
interest has to be rapidly removed and immediately processed:
2. Flash frozen in liquid nitrogen (30-60 sec) and stored at -80°C
3. Flash frozen in liquid nitrogen cooled isopentane and stored at -80°C until sectioning in order to minimize proteolysis and conserve PTMs of peptides and proteins.
4. Small sections can also be frozen using dry ice and ethanol
5. Alternatively, the tissue may be frozen in a mixture of dry ice and hexane at -75°C, embedded in a 2% gel of sodium carboxymethylcellulose (CMC) and stored at -80°C until further use.
6. Embedding in gelatine has been used to facilitate handling of small or fragile samples (e.g., biopsies).
7. The freezing process can lead to sample cracking and fragmentation, as different parts of the tissue cool down at different rates and ice crystals may form. To avoid sample damage, the tissue may be loosely wrapped in aluminum foil and frozen in liquid nitrogen, ethanol, or isopropanol at temperatures below -70 °C by gently lowering the tissue into the liquid over a period of 30-60 s. This preserves the shape of the tissue and also protects the biological tissue components from degradation (Schwartz et al. J Mass Spectr., 2003)
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Effect of keeping the sample in RT
Rapid Heating=treatment untreated Slow warming
Heating 1-5 min
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Heat stabilization
1. Similarly, biopsy/autopsy human material can be stored at -80°C after being subjected to a conductive heat transfer. The methodology was developed to stabilize biological tissues and fluids at the moment of sampling (Denator AB, Gothenburg Sweden)
2. The tissue stabilization system utilizes a combination of heat and pressure under vacuum and its utility was demonstrated by monitoring the PTMs and stability of proteins and by checking the enzymatic activities in the mouse and human brain.
www.denator.com
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Heat stabilization
www.denator.com
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Step 2: sectioning the tissues1) Contamination with embedding media for cryosection, such as agar, a
polysaccharide, Tissue-Tek® and OCT (optimal cutting temperature compound), a combination of polyvinyl alcohol and polyethylene glycol polymers, should be avoided as they suppress ion formation in MALDI MS.
2) To facilitate handling of small or fragile samples (i.e., biopsies), embedding in gelatine or agarose has also been used.
3) At present, the most widely used technique is to affix flash frozen tissue on a cold MALDI target plate or to a conductive surface, i.e. nickel or ITO-coated (indium-tin-oxide) glass slide with a minimal amount of OCT so that it is not in direct contact with the sectioned tissue or microtome blade during sectioning.
4) The microtome blades (preserved in mineral oil) should also be washed with acetone or methanol to prevent chemical contamination if no disposable blades are used.
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OCT effect on IMS spectra
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Matrix Application
• Matrix application is vital for quality image resolution
• Must contact sample as fine, liquid mist
• Current procedure involves manual application with airbrush
Barrett-Wilt, G., USA
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Bruker IMAGE-PREPWith SPR
Nano spotter
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(Immuno)-Histochemistry
Post analysis HE staining
Consecutive sections staining
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Some clinical cases
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Albuminuria and chronic kidney disease
>> Searching for non-immunogenicalbumin peptide fragments
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Analysis of endogenous peptides
Hematoxylin-Eosin Y MW= 2791.0 2 Da±
MW= 2791.0 2 Da±
200 µm
50 µm
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SA-coated slides were imaged within the same day by MALDI-TOF MS using an Autoflex III™ imager mass spectrometer, equipped with Smartbeam™ (Bruker Daltonics, Germany). The lateral resolution for the MALDI imaging was set to 200x200 m for initial tissue screening and 50x50 m for more detailed distribution analysis of MS peaks of interest.
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Post-analysis HE staining
ALb1-24 peptide localizing to the cortex within, and in proximity of tubular cells
Analysis of endogenous peptides
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Comparison of average spectra from selected ROI
ROIX_sectionY= represents overall average spectrum from a given region3 and 4=represents overall average spectrum from a scanned tissue section
ROI1_section3
ROI2_section3
ROI1_section4
Section 3Section 4
m/z= 2791 Da
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Analysis of selected signal on consecutive sections
Selected signal of m/z=2791 Da (yellow)RMR kidney, 10µm thick, SA sprayed
ROI
sections1-5
2791 2 Da
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Neuronal Ceroild Lipofuscinosis (NCL)
>> Searching for changes in severalaccumulating peptide fragments
(misfolding of Saposin)
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Visualization of molecular species allows differential comparison (Ctsd +/+ vs Ctsd +/-)
SA-coated kidney slides were imaged within the same day by MALDI-TOF MS using an Autoflex III™ imager mass spectrometer). The lateral resolution 150 x 150 m for initial tissue screening (measurement range 2-30 kDa). WT-wild type Ctsd +/+, HT- heterozygous Ctsd +/-.
m/z 4767 2 Da
m/z 6662 2 Da
WT (Ctsd)HT (Ctsd)
WT (Ctsd)HT (Ctsd)
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Alzheimer’s disease
Alois Alzheimer
Amyloid protein plaques in the brain
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Alzheimer’s disease Abeta lesions
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Alzheimer’s Amyloid Precursor Protein (APP) processing
sA
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?
Various genetical mutations in amyloid protein lead to different formsof the amyloid protein
Observed by AB are the 1 – 40; 1 – 42In the reality we have 1 minus somethingand 40 plus something!
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MALDI-IMS analyses on the Arctic AD brain
Validation of results
Resolution is 200 x 200 µm, positive linearmode. Scale bar in (a-g) represents 2 mm.
A peptides detected with MALDI-TOF and MALDI-IMS analyses
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Validation of results (IP and MALDI-MS)
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Imaging Mass Spectrometry (IMS) of a Specific Fragment of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase 2 Discriminates Cancer from Uninvolved Prostate TissueLisa H. Cazares,1,2,3 Dean Troyer,1,3 Savvas Mendrinos,3 Raymond A. Lance,3,5, Julius O. Nyalwidhe,1,2,3 Hind A. Beydoun,4 Mary Ann Clements,1,2,3, Richard R. Drake,1,2,3 and O. John Semmes1,2,3
Clin Cancer Res 2009;15(17) September 1, 2009
Diagnostic value?
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Special Applications for IMS
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Multiplex Targeted Secondary Detection(MTSD)
• Targeting molecule of interest with specific antibodies which carry a photocleavable mass tag sensor
• The mass tag sensor is a small molecule of known mass easily detectable by MALDI MS (indirect detection)
• The tag is released just before detection step due to irradiation with MALDI laser (photolabile linker)
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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 in the 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 to Langerhans islets and so the synaptophysin-positive cells. The distribution of synaptophysin positive cells in (A) is very similar to that in (B).
Immuno-IMS
TAMSIM (TArgeted Multiplexed MS Imaging)
Thiery, G., et al., Proteomics 2008
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Thiery et al. Proteomics 2008, 8, 3725–3734
TAG 1 – TAG 1000
With High Resolution Mass Spectrometerswe can have several hundreds of TAG’s
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Resolving power of an MS
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IMS FT-ICR-MS(Fourier Transformation Ion Cyclotron Resonance MS)
Sensitivity in the zmol range
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Limitations, so far …
• IMS Scans are best performing for <30 kDa m/z range
• Sample preparations challenges:• Tissue cryosections (mostly)• Autolysis possible• Tissue fixation• Suppression effect• Reproducibility is a concern
• Interpretation of complex spectra is not straight forward• Protein identifications challenges• Possible displacement or loss of small molecules after
trypsinization for MS/MS
• Minor proteins enrichment is a handicap• Requires other staining methods for comparison
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main funding:
Clinical Proteomics/Mass Spectrometry Unit, Biomedicum Helsinki:Rabah SoliymaniAthanasios Gotsopoulos Maciel LalowskiEvanthia Monogioudi
Haartaman Institute, HelsinkiH. Kalimo
University of Uppsala, SwedenOla PhilipsonLars LannfeltLars Nielsen
Mario Negri Institute for Pharmacological Research, Bergamo, ItalyDaniela MacconiAriela Benigni
University of Helsinki
COST actions:
EuroKUP
IMS Action
Juselius Foundation
EU FP7 DEMCHILD