Pierre Chaurand

Vanderbilt University Medical Center, Nashville, TN.

Molecular Imaging of Tissue Sections by MALDI MS

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Imaging Mass Spectrometry of Thin Tissue Sections

To obtain information on the local protein composition at any coordinate on the section

To reconstruct 2-dimensional density maps (or images) for all of the signals detected

To obtain molecular profiles and images indicative of health status

Discovery tool to answer fundamental questions relevant to protein expression in normal and diseased tissues

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ImagingProfiling Slice frozen tissue on cryostat (~12 μm thick)

Acquire mass spectra

Thaw slice onto MALDI plate, allow to dry

Low Density DropletArray

m/z 18 388

Apply matrix

Laser

+ + +

Molecular profiles

Molecular Images

High Density DropletArray

Laser

+ + +

Spray coating

Laser

+ + +

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Cryostat head Tissue specimen

Cryostat blade Tissue section

Conductive glass slide

Embedding media

2 mm

Section rinsing

Section staining

Optimizing tissue processing protocols

Chaurand et al. J. Proteome Res. 5, 2889-2900 (2006).Copyri

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Unwashed spotted day 1 acq. day 1Unwashed spotted day 1 acq. day 2Unwashed spotted day 2 acq. day 2

Washed spotted day 1 acq. day 1Washed spotted day 1 acq. day 2Washed spotted day 2 acq. day 2

2000 4000 6000 8000 10000 12000 14000 16000 18000

Rel

ativ

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ities

Mass-to-charge (m/z)Erin H. Seeley Copyri

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Shimadzu Chemical Inkjet Printer – Piezo print head

Labcyte Portrait 630 MALDI reagent multi-spotter – Acoustic ejection

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Matrix reservoir

Coupling reservoir

Coupling membrane

yx

Sample plate

Transducer

RF generator

Waveform generator

Tissue section

600 µm 2000 5000 8000 11000 14000 17000Mass-to-charge (m/z)

Rel

ativ

e in

tens

ities

Manually spotted

Automatically spotted

Automated Matrix Deposition

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15000

Tumor (A)

Tumor (B)

Non Tumor (C)

Non Tumor (D)

Mass-to-charge (m/z)4000 8000

x 5

A B

C

D

3 mm

Non-Tumor

Tumor

**

*

**

*

* *

**

*

* *

*

*****

*

*

Rel

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ities

Profiling of a glioma biopsy

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Cut and mount sections onto MALDI plate. Rinse

Apply matrix

Laser

+ + +

Acquire Protein profiles

N N N N T T T T T T T

Pre-processing

Data mining & Classification

Workflow for MALDI MS Profiling of Clinical Biopsies

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MatrixSpotting

MassSpectrometry

HistologicalAnalysis

M3 M2 M1*

Mass (m/z)

Integrated Strategy for Profiling Histo-pathologically Significant Sites

Cornett et al, Mol. Cell Proteomics, 5, 1975-1983 (2006).Copyri

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Histology Directed Matrix Deposition

From this…. Arbitrary large spots

Breast tumor Biopsy:Pathology Coded ID’s

Blue - DCISRed - StromaGreen - Inflammatory CellsBlack - IMC

To this…pathologist-selected features

Cornett et al, Mol. Cell Proteomics, 5, 1975-1983 (2006).Copyri

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DCIS and IMC Exhibit Higher Abundance of m/z 14,098

Average spectrum representation for each annotated cell type

Rel

ativ

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n in

tens

ity

Cornett et al, Mol. Cell Proteomics, 5, 1975-1983 (2006).Copyri

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Matrix printing

Imaging of a Mouse Brain Section

Create custom plate file

1000 μm

m/z 17885 m/z 11839 m/z 20688

m/z 11790 m/z 6759 m/z 7338

0 % 100 %

Automated data acquisition

2014 15579 29144 42708 56273 69838

Mass-to-charge (m/z)

Inte

nsity

Data processing and Image reconstruction

Aerni et al., Anal. Chem. 78, 827-834 (2006).Copyri

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3000 6000 9000 12000 15000 18000Mass (m/z)

Rel

ativ

e In

tens

ities

9939

6651 8124113078367 14006

9328 14855 15830

8454892653064282

1519810812

12435

4965

99768772

6544 12134

15821

85655637

5487

99398928

15198

5031

4422

1200

5

7389

8813

CortexCapsule

Medulla

Calyx

5 mm

Rat Saggital Kidney Section

Printing resolution: 300 µm

Chaurand et al., Curr. Opin. Biotechnol. 17 (2006) 431-436.Copyri

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m/z 11307 - Histone H4

m/z 4422 m/z 6544

m/z 8813

m/z 8565 - Ubiquitin

m/z 15198 - Hemoglobin Alpham/z 12134 - Cytochrome C

m/z 12005

m/z 14855

m/z 8367

m/z 7389

m/z 84540

100%

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m/z 2545.7m/z 2207.3 m/z 2767.3 m/z 3052.3 m/z 3752.6 m/z 3945.4

m/z 4194 m/z 4308 m/z 5173 m/z 5201 m/z 5951 m/z 6244

m/z 6773 m/z 6800 m/z 6979 m/z 7464 m/z 9401 m/z 9419 m/z 9441 m/z 9939

m/z 10104 m/z 10147 m/z 10352 m/z 11160 m/z 10943 m/z 11240 m/z 11634 m/z 13901

m/z 14005 m/z 21239 m/z ~21431m/z 14046 m/z 14090 m/z 21910 m/z ~22111 m/z 22561

Tumor lesion

Biomarker discovery

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Optical image Brain

m/z 20736

White matter

m/z 10271

Corpus Callosum

m/z 5978

Eye socket

m/z 6858

Rat Head Subsection

Whole Rat Sagittal Section Imaging

1 cm

100%100%

50%50%

0% signal 0% signal intensityintensity

Khatib-Shahidi et al., Anal. Chem. 78: 6448-6456 (2006).Copyri

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1900 7520 13140 18760 24380 30000Mass (m/z)

Average Ion Spectrum for Whole Rat Sagittal Section

1 cm

Kidney Cortex m/z 4643Cecum wall m/z 5155Thymus m/z 6893Heart m/z 7921Liver m/z 14321Testis m/z 14400Brain m/z 21952Muscle m/z 11836

Whole Rat Imaging

Khatib-Shahidi et al., Anal. Chem. 78: 6448-6456 (2006).Copyri

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Liver

Kidney

Spleen

Cecum

Stomach

Heart

Lung

1 cm

MALDI MS/MS Image of OLZ (m/z 256) in stomach 2 hours post-dose

Optical image of orally dosed (8 mg/mL) rat abdomen

MS/MS Image of OLZ, 10x signal intensity with stomach signals masked

N

NH S CH3

N

N

CH3

282

256

213

Olanzapine(MW 313)

Inte

nsity

240 250 260 270 280 290Mass (m/z)

282.1

256.1

Sheerin Khatib-Shahidi

Drug Imaging: Olanzapine

0 100%

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kidney liver lung spinal cord brain

testis bladder Spleen thoracic cavity thymus

2h Optical Image

Olanzapine m/z 313→256

N-desmethyl metabolite m/z 299→256

2-hydroxymethyl metabolite m/z 329→272

fur

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3000 6000 9000 12000 15000 18000Mass (m/z)

Inte

nsity

4964

5487

56378568

6721 14135

9983

6651 812699436278 7539 106608369 8931 1520511315

1214015863

Myelin basic protein(MBP, MW 14134)

On-tissue trypsin digestion: Rat brainSection 1: IMS of proteinsMatrix printing - SA - Shimadzu CHIPData acquisition - Bruker Ultraflex II

Reid Groseclose et Al, J. Mass Spectrom. 42, 254-262 (2007).

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500 900 1300 1700 2100 2500Mass (m/z)

Inte

nsity

842.53

616.21870.56

734.59

679.52

1198.68709.40

1460.65

1496.75

944.551131.57856.54

762.61

1388.68566.41 1801.74

1090.582141.931694.67

1924.742229.92

On-tissue trypsin digestion: Rat brainSection 2: IMS of tryptic peptidesDigestion with trypsin - Shimadzu CHIPMatrix printing - 2,5-DHBA – Shimadzu CHIPData acquisition - Bruker Ultraflex II (rTOF) MBP

m/z 699.47 m/z 726.47 m/z 1067.60 m/z 1100.12

m/z 1131.84 m/z 1336.63

m/z 1460.99 m/z 1502.90

Reid Groseclose et Al, J. Mass Spectrom. 42, 254-262 (2007).Copyri

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Reid Groseclose

On-tissue trypsin digestion: Rat brainSection 2: MALDI MS/MS of tryptic peptides (m/z 1067.60)

1025.9

b7

c6a8

50 256 462 668 874 1080Mass (m/z)

Inte

nsity

y2

y3W

Rb4y1 y4

y3

403.1

y2

b5

b8+H2O

b8b7

1008.0

b9-17

[M+H]+

b6 b7-17

b8-17

b6

F-S-W-G-G-R-D-S-Ry1y2

b8b7

y3

b6

y4

b5b4

x5

Bruker Ultraflex II

Shimadzu QIT b8+H2O

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a. intra-section registration

b.

D

V

P

A

cx

hicm

SN

3D-Imaging MS

3D reconstruction ofthe substantia nigrain the mouse brain

M. Andersson et al, Accepted in Nature Methods, 2007Copyri

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Substance P(m/z 1348)

PEP-19(m/z 6718)

3D Imaging Mass SpectrometryA.

B.

2 mm

0 %

100 %

0.0 mm

+0.8 mm

+1.6 mm

+0.6 mm

+1.4 mm

+2.4 mm

+0.2 mm

+1.0 mm

+1.8 mm

+0.4 mm

+1.2 mm

+2.0 mm

0 %

100 %

0.0 mm

+0.8 mm

+1.6 mm

+0.6 mm

+1.4 mm

+2.4 mm

+0.2 mm

+1.0 mm

+1.8 mm

+0.4 mm

+1.2 mm

+2.0 mminterpeduncular nucleus

M. Andersson et al, Accepted in Nature Methods, 2007Copyri

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i=0 i=1 i=2 i=3 i=N……

ReconstructedBlockface Volume

r1

r2

r3

rN

Integrating IMS and MRIStep 1: reconstruction of an optical blockface volumeEach image in the blockface volume is registered to a previously registered image in the volume

T. Sinha, S. Khatib-Shahidi et al. Accepted in Nature Methods (2007). Copyri

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Integrating IMS and MRIStep 2: Insertion of ion images into reconstructed blockface volume

m/z 14182

m/z 5826

T. Sinha, S. Khatib-Shahidi et al. Accepted in Nature Methods (2007). Copyri

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Integrating IMS and MRIStep 3: Co-registration of MR to blockface images

T1

T2 ADC

T. Sinha, S. Khatib-Shahidi et al. Accepted in Nature Methods (2007). Copyri

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Areas of Further Development

Increase number of proteins analyzed to >>1000Develop sample protocols to investigate peptidesAchieve high sensitivity above 50 kDa Integrate detergents into tissue protocol for membrane associated proteins

Pixel-to-pixel cycle time of about 100 ms needed

Integrate bioinformatic tools, image processing

Improve protein identification protocols

Develop sample protocols targeted at specific tissues/diseases

Develop sample protocols to investigate FFPE biopsies

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Conclusions

Excellent discovery tool

MW annotated patterns keyed to tissue location

Augments (not replaces) current molecular technologies

Significant clinical potential (disease diagnosis, state and progression, prognosis, risk assessment?)

Provides temporal proteomic profile that, with genomic profile, will be vital to personalized medicine

Tissue Profiling/Imaging MS

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FundingNIH (NCI, GMS, NIDA)DODVanderbilt University

Acknowledgments

OthersPer Andren, Uppsala UniversityAlain Brunelle, CNRS-Gif-sur-Yvette Walter Korfmacher, Schering-PloughMarkus Stoeckli, Novartis

Vanderbilt CollaboratorsCarlos ArteagaDavid CarboneRobert Coffey Jr.Marie-Claire Orgebin-CristAriel DeutchPierre MassionPat Levitt Robert MatusikHal MosesJennifer PietenpolNed Porter Yu ShyrSusan KasperKen Schriver

MSRCRichard Caprioli (Director)Shannon CornettMichelle ReyzerErin SeeleyJoey LathamStacey OppenheimerSheerin Khatib-ShahidKristin BurnumKristen HerringSatu PuolitaivalHans Reudi AerniReid GrosecloseWilliam HardestyWhitney Ridenour

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