Molecular imaging and metabolite profiling of
tissues sections by FTICR-MS MALDI and LESA
C. Logan Mackay
SIRCAMS
School of Chemistry
University of Edinburgh
Edinburgh
UK
Richard Goodwin
DMPK
R&D
AstraZeneca
Södertälje
Sweden
MS Imaging - Small molecules
Aim
Molecular imaging - unlabelled
Problem
Metabolites missing
Solution
LESA profiling
Summary
Ultraflex eXtreme
TOF/TOF
Ultraflex II
TOF/TOF
Synapt G2
12T FT-ICR-MS
LESA
Liquid extraction surface analysis
Raclopride (a dopamine D2 receptor antagonist)
C15H20Cl2N2O3 [M+H] m/z 347.0924
Organs:
Dose:
Time:
Matrix:
Application:
Brain
Liver
Kidney
Whole body
7.5
2 mg/kg
1
5
30 m.a.d
α-Cyano-4-hydroxycinnamic acid
solvent free dry matrix
“Qualitative and quantitative MALDI imaging of the positron emission tomography ligands raclopride (a D2 dopamine antagonist) and SCH
23390 (a D1 dopamine antagonist) in rat brain tissue sections using a solvent-free dry matrix application method.” Anal. Chem.
15;83(24):9694-9701 Goodwin RJ, Mackay CL, Nilsson A, Harrison DJ, Farde L, Andren PE, Iverson SL (2011)
Raclopride 2 mg/kg 1 min post i.v
Synapt MS
Synapt MS/MS
MALDI MS imaging analysis of neurological
positron emission tomography (PET)
ligands: application to both established and
developmental compounds
Tue: 2.20pm
Synapt MS/MS
14C
Quantitative Whole Body Autoradiography (QWBA)
MS imaging
Stomach Liver Heart Spinal cord
FT-ICR MS
350 µm
1 minute after dose
1 mg/kg
Brain
14C
Quantitative Whole Body Autoradiography (QWBA)
Raclopride 2 mg/kg
1
5 30 min Vehicle
Raclopride 2 mg/kg
1 min post i.v
10.9 ± 0.6 nmol/g
Cortex 12.9 ± 0.3 nmol/g
30 min post i.v
100 %
1.7 ± 0.1 nmol/g
Relative abundance 15% after 30
Cortex 5.4 ± 0.2 nmol/g
Relative abundance 40% after 30
1 min
Cortex
Outer Medulla
Inner Medulla
30 min
100
42%
100
41%
100
46%
MS Imaging
Raclopride 2 mg/kg
1 min post i.v
2mg/kg
7.5mg/kg
10.9 ± 0.6 nmol/g
30 min post i.v
100 %
1.7 ± 0.1 nmol/g
Relative abundance 15% after 30
m/z 347 (Raclopride)
1 m.a.d.
30 m.a.d.
100% (5.1 a.u)
22.4% (1.1 a.u)
100% (16.1 a.u)
21.1% (3.4 a.u)
Conclusion
1. Linear response between dose and
detection by MSI
2. In brain - correlation between MSI
relative abundance and
autoradiography abundance but not in
kidney and liver
30 minute after dose
2 mg/kg
RT: 5.09 - 17.96
100
95
90
NL: 5.14E6
Base Peak F: FTMS
+ c ESI Full ms
[200.00-700.00] MS
Racloprid30min382d
il_MDF
13.46
Plasma
30 m.a.d.
85
Raclopride
80
75
70
M4
65
60
12.30
55
M1 m/z 381
M2 m/z 523
M3 m/z 409
M4 m/z 333
50
45
40
35
M1
M2
M3
Raclopride
m/z 347
30
25
20
15
8.10 8.58
16.77
11.90
10
6.81
11.72
8.96
5
5.72
7.27
9.09
0
6
8
10
10.93
12
Time (min)
13.65 14.94
16.20
14
16
Unable to detect any metabolites by MALDI
MSI
30 minute after dose
2 mg/kg
RT: 5.09 - 17.96
100
95
90
NL: 5.14E6
Base Peak F: FTMS
+ c ESI Full ms
[200.00-700.00] MS
Racloprid30min382d
il_MDF
13.46
Plasma
30 m.a.d.
85
Raclopride
80
75
70
M4
65
60
12.30
55
M1 m/z 381
M2 m/z 523
M3 m/z 409
M4 m/z 333
50
45
40
35
M1
M2
M3
Raclopride
m/z 347
30
25
20
15
8.10 8.58
16.77
11.90
10
6.81
11.72
8.96
5
5.72
7.27
9.09
0
6
8
10
10.93
12
Time (min)
13.65 14.94
16.20
14
16
Unable to detect any metabolites by MALDI
MSI
LESA
Liquid
Extraction
Surface
Analysis
700 nL
MeOH/H2O
(0.1 formic)
Quad isolation (m/z 400 ± 175)
Intens.
x108
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
347.09
Measured
1.5
349.09
1.0
0.5
348.07
348.93
350.09
351.09
0.0
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C15H20Cl2N2O3, M+nH ,347.09
347.09
5000
Theoretical
4000
349.09
3000
2000
1000
348.10
350.09
30 minute after dose
Raclopride 2 mg/kg
351.09
0
347
348
349
350
351
352
353
Contribution to
QWBA abundance
Name
Measured
Theoretical
Formula
Raclopride
m/z 347.0922
m/z 347.0924
C15H20Cl 2N2O3
QWBA
labeled
M1
m/z 381.0519
m/z 381.0518
M2
m/z 523.1245
m/z 523.1246
C13H17ClN2O7S C21H28Cl 2N2O9
labeled
labeled
M3
m/z 409.0814
m/z 409.0831
M4
m/z 333.0751
m/z 333.07672
C15H21ClN2O7S C14H18Cl 2N2O3
labeled
unlabeled
m/z
Intens.
x107
Intens.
x107
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
Measured
0.8
381.89
0.8
0.6
0.4
0.6
383.03
381.01
381.08
m/z 381.0111
m/z 381.0794
0.2
381.0111
0.4
381.0794
0.0
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C13H17ClN2O7S, M+nH ,381.05
381.05
0.2
5000
Measured isotope distribution
Theoretical
4000
0.0
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C13H17ClN2O7S, M+nH ,381.05
381.0518
3000
5000
m/z 381.0518
2000
383.05
1000
4000
382.06
0
381.00
381.25
381.50
381.75
382.00
382.25
382.50
382.75
383.00
3000
2000
Theoretical isotope distribution
1000
0
381.00
381.02
Name
Measured
Theoretical
Formula
Raclopride
m/z 347.0922
m/z 347.0924
C15H20Cl 2N2O3
QWBA
labeled
381.04
381.06
M1
m/z 381.0519
m/z 381.0518
381.08
381.10
M2
m/z 523.1245
m/z 523.1246
C13H17ClN2O7S C21H28Cl 2N2O9
labeled
labeled
381.12
381.14
M3
m/z 409.0814
m/z 409.0831
381.16
m/z
M4
m/z 333.0751
m/z 333.07672
C15H21ClN2O7S C14H18Cl 2N2O3
labeled
unlabeled
Contribution to
QWBA abundance
m/z
Intens.
x107
glass kidney 2mg_kg cortex_000001.d: +MS
524.274
8
Measured
6
527.176
523.124
4
526.242
525.119
2
527.481
525.282
523.301 523.539
528.185
524.115
527.958
524.543
0
glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.13
523.124
5000
Theoretical
4000
525.122
3000
2000
524.128
1000
526.125
527.119
528.122
30 minute after dose
Raclopride 2 mg/kg
Name
Measured
Theoretical
Formula
Raclopride
m/z 347.0922
m/z 347.0924
C15H20Cl 2N2O3
QWBA
labeled
M1
m/z 381.0519
m/z 381.0518
M2
m/z 523.1245
m/z 523.1246
C13H17ClN2O7S C21H28Cl 2N2O9
labeled
labeled
0
523
M3
m/z 409.0814
m/z 409.0831
M4
m/z 333.0751
m/z 333.07672
C15H21ClN2O7S C14H18Cl 2N2O3
labeled
unlabeled
524
525
526
527
528
m/z
Intens.
x107
glass kidney 2mg_kg cortex_000001.d: +MS
524.274
8
Measured
6
Intens.
x107
527.176
glass kidney 2mg_kg cortex_000001.d: +MS
523.124
523
4
m/z 526.1246
4
526.242
525.119
2
3
527.481
525.282
523.301 523.539
528.185
524.115
527.958
524.543
0
glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.13
523.124
2
5000
Measured isotope distribution
1
Theoretical
4000
525.122
3000
0
glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.13
523
2000
m/z 526.1245
5000
524.128
1000
526.125
527.119
4000
528.122
0
523
3000
2000
Theoretical isotope distribution
1000
0
523.06
523.08
Name
Measured
Theoretical
Formula
Raclopride
m/z 347.0922
m/z 347.0924
C15H20Cl 2N2O3
QWBA
labeled
523.10
523.12
M1
m/z 381.0519
m/z 381.0518
523.14
523.16
M2
m/z 523.1245
m/z 523.1246
C13H17ClN2O7S C21H28Cl 2N2O9
labeled
labeled
523.18
M3
m/z 409.0814
m/z 409.0831
523.20
m/z
M4
m/z 333.0751
m/z 333.07672
C15H21ClN2O7S C14H18Cl 2N2O3
labeled
unlabeled
524
525
526
527
528
m/z
N=7
S.D 7%
30 minute after dose
Raclopride 2 mg/kg
Name
Measured
Theoretical
Formula
Raclopride
m/z 347.0922
m/z 347.0924
C15H20Cl 2N2O3
QWBA
labeled
M1
m/z 381.0519
m/z 381.0518
M2
m/z 523.1245
m/z 523.1246
C13H17ClN2O7S C21H28Cl 2N2O9
labeled
labeled
M3
m/z 409.0814
m/z 409.0831
M4
m/z 333.0751
m/z 333.07672
C15H21ClN2O7S C14H18Cl 2N2O3
labeled
unlabeled
S.D 56%
QWBA 30 minute after dose
Raclopride 2 mg/kg
MSI
Dissected kidney 30 minute after
dose raclopride 2 mg/kg
m/z 347
Summary
Whole body
ESI LESA
profiling
Fast
MALDI MS
imaging
Cheap
QWBA
Effective
Tissue specific
Study
Relative
distribution
Absolute Quant.
Label available
Time scale
&
Scope of analysis
Acknowledgments
University of Edinburgh (SIRCAMS)
Pat Langridge-Smith - Oral - Tue 9:10 (Room 118-120)
David Clarke
- Oral – Wed 9:30 (Exhibit hall A)
Andrew Dennison
- Oral – Tue 3:30 (Ballroom CD)
Uppsala University
Per Andren
Anna Nilsson
- Oral – Tue 2:50 (Ballroom B)
AstraZeneca
Advion
Suzanne Iverson
Goran Eklund
Daniel Borg
Mark Allen (UK)
Mark Baumert (UK)