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