Oak Ridge Conference

Imaging Mass Spectrometry – A Deeper View of Biology

Erin H SeeleyMass Spectrometry Research Center

Vanderbilt UniversityApril 19, 2012

Imaging/Profiling: MALDI TOF, TOF/TOF, LTQ, IM QTOF, FTICR( Bruker TOFs, 9.4T FTICR; Thermo LTQ; Waters IM QTOF; AB TOF )

MALDI Mass Spectrometry Technology

Laser Detector

Sample Plate

MALDI -TOF MS (linear)

Whole Body Drug and Metabolite Imaging

Sheerin Khatib-Shahidi et al.

Whole Body Protein Imaging•Correlate protein expression changes with drug distribution

Sheerin Khatib-Shahidi et al.

Phospholipid Imaging

Kristin Burnum et al.

In Situ Proteomics (Peptide Imaging)•In situ enzymatic digestion prior to IMS analysis

Reid Groseclose et al.

3D Protein Imaging of a Glioma Tumor Mouse Brain•Integrated 3D IMS with in vivo MRI

Tuhin K Sinha et al.

Imaging Mass Spectrometry (IMS) can visualize the spatial localization of numerous

biomolecules directly from tissue sections without any prior knowledge of the specific molecules being analyzed

Combine Phospholipid and Proteins imaging to Visualize Molecular Changes during Embryo Implantation

Kristin Burnum et al.

MALDI Imaging

Molecular images

Section tissue & apply matrix

Raster of tissue

m/z

Laser

Ion images from a single raster of a mouse brain section– each image is of a different m/z (this set ~1/10 of data set)

MALDI MS Profiling(Histology Directed Molecular Analysis)

sectionHistology & targetedmatrix spotting

MALDI MS of targeted areas

5000 10,0000

rela

tive

inte

nsity

m/z

striatumcortex

Workflow for Histology Directed MS Profiling

Tissue section on MALDI target

Histological areas marked by a biologist / pathologist

1 cm

Coordinates transferred to instrumentation

Matrix deposited on tissue at points of interest from histology

1 mm

5000 10000 15000

0

1000

2000

3000

4000

Inte

nsity

m /z

normal lobular units normal stroma tumor stroma DCIS IMC

Mass spectra from profiled spots

Melinda Sanders

Correlating Histology and MS

-- Fresh frozen tissue

-- Formalin fixed paraffin embedded

Reid Groseclose

FFPE Mouse Brain

Serial Tissues SectionsMounted onto MALDI targets

Antigen Retrieval

On-Tissue TrypsinDigestion

MALDI Imaging /Profiling Mass Spectrometry

Working with Formalin Fixed Paraffin Embedded Tissue

Spitz nevus

Spitzoid Melanoma

Lymph Node with metastasis

Rossitza Lazova (Yale)

MS Analysis of Spitzoid Lesions in FFPE Biopsies

Training set # Patients Classification Accuracy (%)

Spitz nevi (SN) 26 100

Spitzoid Malignant Melanoma (SMM)

25 96

Classification of Spitzoid Lesions based on a 5 peptide feature signature

Validation (test) set # Patients Classification Accuracy (%)

Spitz nevi (SN) 30 97

Spitzoid Malignant Melanoma (SMM)

29 90

56 SN and 54 SMM from Yale University Spitzoid Neoplasm Repository

Melanoma

Spitz Nevus0

20

40

60

80

Inte

ns. [

a.u.

]

800 900 1000 1100 1200 1300 1400 1500m/z

SpitzoidMelanomaSpitz Nevi

HighGrade

LowGrade

5 mm

m/z 8451

m/z 5171

Soft Tissue Sarcomasfrozen tissue

Ginger Holt

High Grade STS; Tumor Necrosis; Normal Tissue Adjacent to High Grade STS; Low Grade STS; Normal Tissue Adjacent to Low Grade STS

Protein Class Imaging Results

m/z 4747

Distance from Margin (µm)

Rel

ativ

e In

tens

ity

HG TumorNormal

10000-5000 5000-10000

Histological Margin

protein

lipidm/z 725

LG TumorHG TumorNormal

Normal

NORMAL

Histology margin

Feature amplitude

Relative Protein Distributions

Potential molecular margin

TUMOR

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A

B

C

D

EF

G

H

IJ

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A

B

C

D

E

F

G

H

I

J

Mass Spectrometry Class Image12 peptide signature Histological Designation

Clear Cell Renal Cell CarcinomaFFPE TMA

TumorNormal

Todd Morgan, Peter Clark

Internal classification accuracy = 99%External validation accuracy = 90%

Profiling / Imaging of Drugs, Drug Metabolitesand Other Small molecules

Imaging Low MW Molecules

250 300 350 400 450 500 550 600m/z

1

2

3

5x10

• MS/MS (e.g.: LTQ, TOF/TOF)• High resolution (e.g.: FTICR, Orbitrap, TOF)• Ion mobility (e.g.: QqTOF)

Principle of CASI (continuous accumulation of selected ions) to enhance dynamic range

courtesy Bruker application note FTMS-37

StorageHexapole

Mass spectrum from lung tissue biopsy obtained on a low res MS (LTQ)

Study of Rifampicin Treated Rabbit Infected with TB

Quad Selection mass: 821.5Quad window: 7 DaCASI fills: 300

Spectrum from Rif Treated TB Lung Tissue by FT-ICR MS (CASI)

Microscopic image

Matrixm/z 821.169

Rifampicinm/z 821.401Accuracy: 3 ppm

Bacterial Lipid*PI(33:1)m/z 821.521Accuracy: 2.7 ppm

LipidPG(40:6)m/z 821.536Accuracy: 2.2 ppm

*predicted Mycobacterium tuberculosis lipid from the species-specific LipidMaps Database.

New Technology Initiatives

Increased Imaging Speed (<< 1 sec/pixel)

High Spatial Resolution Imaging (1-2 μm)

Increased Sensitivity – Derivatization and Targeted Analyses

Ease of Use: Matrix Pre-Coated Targets

MALDI (TOF) IMS using a 5 kHz Nd: YAG continuous laser

m/z 734.4 ( PC 32:0)m/z 788.5 (PC 36:1)m/z 806.5 (PC 38:6)

lipid images acquired at a rate of 30 pixels/s Total image time: < 10 min

Jeff Spraggins

Yellow – m/z 5020; Blue – m/z 8396; White – m/z 8441; Magenta – m/z 8776; Red – m/z 10165; Purple – m/z 10883; Green – m/z 11838; Pink – m/z 21010

3D volume of m/z 5020alpha globing residues 2-47

Perspectives

Instrumental / Methodology Challenges and Needs

Sensitivity - achieve more global coverage (fraction of proteome now observed)

Resolution - better lateral resolution (routine single cell imaging)- higher MS resolution (better resolve isoforms, PTMs, isobars)

Mass Range - routinely beyond 100 kd

Identification - in situ - fast, simple, accurate

Quantitation - reagents and methods - isotope based, relative and absolute

Validation - cross-lab (std protocols), cross-platform reproducibility/standardization

Availability - single manufacturer must provide entire technology ‘solution’

Major Advantages of Direct Tissue Analysis by MS

Molecular images and patterns easily correlate with histopathology

Native molecular distributions provide new biological insights

Excellent discovery technology (no target specific reagents needed)

MS gives exceptionally high throughput (seconds per sample)

Multiple images produced simultaneously at discrete MW values

Mass SpectrometryResearch Center

Richard CaprioliMichelle ReyzerKen ShriverAndrey ZavalinJeff SpragginsPeggi AngelHans Reudolf AerniSara FrappierLisa ManierEduardo DiasJunhai YangKerri GroveJamie AllenBao TranJosh NicklayKristie RoseGlenn Harris

Vanderbilt Collaborators

David HacheyKevin ScheyPaul LaibinisJohn GorePierre MassionEric SkaarReid ThompsonBilly HudsonJohn OatesOlivier BoutaudNancy BrownMark MagnusonRandy BlakelyAnna CarneiroAriel DeutchLynn MatrisianRay Mernaugh

FundingNIH (GMS, NCI, NCRR)Department of DefenseThe Robert and Helen Kleberg

FoundationThe T.J. Martell FoundationThe Gates Foundation

OthersPeter Wild, U ZurichAlan Soloman , U TennesseeJohn Mayer, HarvardReid Groseclose, GlazoSmithKlineKristin Herring, Dept of DefenseKristin Burnham, Batelle PNW LabsPierre Chaurand, U MontrealShannon Cornett, Bruker DaltonicsSK Dey, U CincinnatiGiovanni Sindona, U CalabriaAmosy M’Koma, MeharryGwendoline Thiery, HarvardKristina Schwamborn, Univ. Munich