high-resolution maldi-ft-icr ms imaging for the in-situ ... · aichler et al., angew chem int ed...

High-resolution MALDI-FT-ICR MS Imaging

for the in-situ Analysis of Metabolites

from Intact Tissues

Axel Walch

Research Unit Analytical Pathology

Neuherberg, 2016-10-12

Balluff et al., Gastroenterology 2012 Aichler et al., Lab Invest 2015 Lahiri et al., Expert Rev Proteomics 2016

Molecular Tissue Analysis by MALDI Imaging Mass Spectrometry

Patient

Animal

Tissue sample

Cell type specific molecular patterns:

Endogen

• Proteome • PTMs • Histone Modifications • Peptidome • Lipidome • Cell metabolism • Hormones • Amino Acids • …

Exogen

• Drugs / Metabolites • Tracer & Contrast Agents • Toxins • …

Stained tissue section

5 mm

Acq

uis

itio

n y

Matrix spotted section

Acquisition x

Average mass spectrum

y

m/z

x

a.u.

5000 10000 15000 20000

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

*

*

*

* *

m/z

UV-Laser

MALDI-TOF MS

H&E staining

Aichler et al., Angew Chem Int Ed Engl 2015 Aichler et al., Lab Invest 2015 Hermann et al., Nat Rev Hepatol Gastroenterol 2009 Rauser et al., Expert Rev Proteomics 2010

Principle of MALDI Imaging Mass Spectrometry („MALDI Imaging“)

MALDI FT-ICR MS imaging of

Drugs and Drug-Related Metabolites

Quantification of Irinotecan / SN-38 by using an Isotope Labeled Compound

Dosed tissue section – dilution series Dosed mouse

ILC & Matrix coverage

(ILC = isotope labeled

compound; d10-Irinotecan)

Quantification

MALDI MS image – drug distribution

Normalization of MS signals: I(A)normalized =

I(A)

I(ILC)

200.0

400.0

600.0

800.0

1000.0

1200.0

0.0

Concentration (pmol/mm²)

R²=0.9951

0 10 20 30 40

Calibration curve of Irinotecan/ILC

2.0

4.0

6.0

8.0

10.0

12.0

0.0

Mean

in

ten

sit

y o

f S

N-3

8

Concentration (pmol/mm²)

R²=0.9546

0 1 2 3 4

Calibration curve of SN-38/ILC

y = ax + b

Irinotecan SN-38

Buck et al., Anal Bioanal Chem 2014

100 mg/kg Irinotecan i.v. 1h after administration

2 mm

Whole Body Drug Distribution and Quantification of Irinotecan / SN-38

H&E

qMSI of Irinotecan and SN-38 on single organs and body fluids

Buck et al., Anal Bioanal Chem 2014

MALDI Imaging of exogenous molecules: Drugs and related metabolites

Grüner et al., Mol Cancer Ther, 2016 Huber et al., Anal Chem 2014 Buck et al., Bioanl Chem 2014 Buck et al., Bioanalysis 2014 Huber et al., Histochem Cell Biol 2014

Pharmacokinetics of Pirfenidone and Related Metabolites

Sun et al., Histochem Cell Biol 2015

Sun et al., Histochem Cell Biol 2015

Pharmacometabolomics of Pirfenidone

Spatially resolved quantification of gadolinium(III)-based magnetic resonance agents by MALDI imaging after in vivo MRI.

B

A

1000 µm 1000 µm

0%

60%

0,00

0,01

0,02

0,03

0,04

0,05

5 min 10 min 30 min 60 min 6 h 24 h 48 h

Infarct Myocardium

Gad

ofl

uo

rin

e M

co

nce

ntr

atio

n [

mM

]

0.00

0.01

0.02

0.03

0.04

0.05

m/z [Da]

rela

tive i

nte

nsit

y

Gadofluorine M

m/z 1527.7

C

Gadofluorine M m/z 1527.7

Aichler et al., Angew Chem Int Ed Engl. 2015

High-resolution MALDI-FT-ICR MS Imaging for the Analysis of

Metabolites from Formalin-Fixed, Paraffin-Embedded (FFPE)

Clinical Tissue Samples

Most Important Clinical FFPE Tissue Categories

In-situ Metabolomics of FFPE Tissues - Experimental Design

1. Sample preparation 2. MSI analysis 3. Data analysis

normal

diseased

Identification &

Pathway analysis (KEGG)

no

rmal

dis

eased

Heatmap & Clustering

Spectra analysis

Average mass spectrum

100 200 300 400 500 600 700 800 900 m/z 0

200

400

600

a.u.

*

* *

*

* * *

Ly & Buck et al., Nat Protoc 2016 Buck et al., Anal Chem 2016 Buck & Ly et al., J Pathol 2015

In-situ Metabolomics : Doing less is more

In-situ Proteomics:

Established protocols for peptide imaging of FFPE tissue:

Xylene 1 Xylene 2 Iso-propanol

100% EtOH

90% EtOH

70% EtOH

50% EtOH

dH2O Citrate buffer

dH2O

Deparaffinization Rehydration Washing AG retrieval

Deparaffinization

Xylene 1 Xylene 2

In-situ Metabolomics:

Protocol for metabolite imaging of FFPE tissues

Tryptic digestion

MALDI MSI

MALDI-FT-ICR MSI

metabolite loss

metabolite loss

Chemical Conservation of Metabolites in FFPE Tissues

239 1226

Fresh-frozen

244

FFPE

72% amongst 1700 m/z species were detected

in two types of specimens

N = 102

75% of identified compounds were detected in all

three types of specimens

N = 4

2 mm

2 mm

1 mm

Fresh Frozen FFPE FFPE TMA

Spatial Conservation of Metabolites in FFPE-Tissues

m/

z 2

59

.01

40

G

ala

cto

se 1

-ph

osp

hate

50

5

200 µm

Buck et al., J Pathol 2015

Colon Cancer Tissues

Spatial Conservation of Metabolites in FFPE-Tissues

50

5

m/

z 2

59

.02

30

H

exo

se 6

-ph

osp

hate

2 mm

2 mm

1 mm

Fresh Frozen FFPE FFPE TMA

200 µm

Buck et al., J Pathol 2015

Colon Cancer Tissues

2 mm

2 mm

1 mm

Fresh Frozen FFPE FFPE TMA

Spatial Conservation of Metabolites in FFPE-Tissues

50

5

m/

z 3

00

.04

00

N

-Acety

lglu

co

sam

ine s

ulf

ate

200 µm

Buck et al., J Pathol 2015

Colon Cancer Tissues

Metabolite Imaging of FFPE samples – Biomedical Applications

• Can this method be used to distinguish between healthy and tumour tissue?

Buck et al., J Pathol 2015

Metabolite Imaging of FFPE samples – Biomedical Applications

• Chromophobe Renal Carcinoma (ChRCC) and Oncocytoma

• Same origin and similar morphology, but different outcome

• Tumours can be distinguished from each other based on metabolic profile

0

Onco ChRCC Oncocytoma ChRCC

0

50

50

2 mm

2 mm

m/z 345.0720

*

m/z 862.6105

*

Buck et al., J Pathol 2015

Metabolites as New Prognostic Marker in Barrett’s Cancer

Covariate Hazard rate P

m/z 256.9975 1.65 0.034

Tumor size (pT) 1.43 0.200

Nodal status (pN) 1.11 0.810

Metastasis (M) 6.19 0.003

0 50 100 150

0.0

0.2

0.4

0.6

0.8

1.0

m/z 256.9975 (cut-off 50% peak intensity)

Disease free survival time [months]

Surv

ival pro

bability

Log rank P = 0.00154

Good prognosis group (n=36) / low mass intensity

Poor prognosis group (n=17) / high mass intensity

High mass intensity / poor prognosis patient:

100 µm

Low mass intensity / good prognosis patient:

100 µm

30

0

30

0

Buck et al., J Pathol 2015

Conclusions

• Metabolite content in FFPE Tissues:

• Can be reliably measured by high mass (and spatial) resolution MALDI Imaging

• Appears to be more robust than other molecules in FFPE tissues

• Large number of molecules can be detected

• Mass resolution and accuracy allow a better annotation, which is limited by MALDI TOF Imaging (metabolites, proteins)

• Can improve molecular tissue diagnostics and tissue based research