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Molecular imaging and metabolite profiling of tissues sections by FTICR-MS MALDI and LESA. C. Logan MackayRichard Goodwin SIRCAMSDMPK School of ChemistryR&D University of EdinburghAstraZeneca EdinburghSödertälje UKSweden. MS Imaging - Small molecules. Aim. - PowerPoint PPT Presentation
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Molecular imaging and metabolite profiling of tissues sections by FTICR-MS MALDI and LESA
C. Logan Mackay Richard GoodwinSIRCAMS DMPKSchool of Chemistry R&DUniversity of Edinburgh AstraZeneca Edinburgh SödertäljeUK Sweden
Aim Molecular imaging - unlabelled
Problem Metabolites missing
Solution LESA profiling
MS Imaging - Small molecules
Summary
Raclopride (a dopamine D2 receptor antagonist)C15H20Cl2N2O3 [M+H] m/z 347.0924
Organs: Brain Liver Kidney Whole bodyDose: 7.5 2 mg/kgTime: 1 5 30 m.a.dMatrix: α-Cyano-4-hydroxycinnamic acid Application: 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)
Ultraflex eXtreme Ultraflex II Synapt G2 12T FT-ICR-MS LESA TOF/TOF TOF/TOF Liquid extraction surface analysis
Synapt MS/MS Synapt MS/MS
Synapt MS
Raclopride 2 mg/kg 1 min post i.v
MALDI MS imaging analysis of neurological positron emission tomography (PET) ligands: application to both established and developmental compounds Tue: 2.20pm
1 minute after dose1 mg/kg
BrainLiver HeartStomach Spinal cord
FT-ICR MS 350 µm
Quantitative Whole Body Autoradiography (QWBA)14C
MS imaging
1 5 30 min Vehicle
100 %
1 min 30 minCortex 100 42%Outer Medulla 100 41%Inner Medulla 100 46%
Raclopride 2 mg/kg
1 min post i.v
30 min post i.v
Raclopride 2 mg/kg
Quantitative Whole Body Autoradiography (QWBA)
10.9 ± 0.6 nmol/g
1.7 ± 0.1 nmol/g
Relative abundance 15% after 30
Cortex 12.9 ± 0.3 nmol/g
Cortex 5.4 ± 0.2 nmol/g
Relative abundance 40% after 30
14C
m/z 347 (Raclopride)1 m.a.d. 30 m.a.d.
2mg/kg 100% (5.1 a.u) 22.4% (1.1 a.u)7.5mg/kg 100% (16.1 a.u) 21.1% (3.4 a.u)
MS Imaging
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
100 %
Raclopride 2 mg/kg
10.9 ± 0.6 nmol/g
1.7 ± 0.1 nmol/g
Relative abundance 15% after 30
1 min post i.v
30 min post i.v
RT: 5.09 - 17.96
6 8 10 12 14 16Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
13.46
12.30
8.10 8.5816.77
11.906.81
11.728.96
13.655.72 14.9410.937.27 16.209.09
NL: 5.14E6Base Peak F: FTMS + c ESI Full ms [200.00-700.00] MS Racloprid30min382dil_MDF
Plasma30 m.a.d.
M4
M1 M2 M3
Raclopride
M1 m/z 381M2 m/z 523M3 m/z 409M4 m/z 333
Raclopride m/z 347
30 minute after dose2 mg/kg
Unable to detect any metabolites by MALDI MSI
RT: 5.09 - 17.96
6 8 10 12 14 16Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
13.46
12.30
8.10 8.5816.77
11.906.81
11.728.96
13.655.72 14.9410.937.27 16.209.09
NL: 5.14E6Base Peak F: FTMS + c ESI Full ms [200.00-700.00] MS Racloprid30min382dil_MDF
Plasma30 m.a.d.
M4
M1 M2 M3
Raclopride
M1 m/z 381M2 m/z 523M3 m/z 409M4 m/z 333
Raclopride m/z 347
30 minute after dose2 mg/kg
Unable to detect any metabolites by MALDI MSI
LESALiquidExtractionSurface Analysis
700 nLMeOH/H2O(0.1 formic)
Quad isolation (m/z 400 ± 175)
30 minute after doseRaclopride 2 mg/kg
Name Raclopride M1 M2 M3 M4 Measured m/z 347.0922 m/z 381.0519 m/z 523.1245 m/z 409.0814 m/z 333.0751Theoretical m/z 347.0924 m/z 381.0518 m/z 523.1246 m/z 409.0831 m/z 333.07672Formula C15H20Cl2N2O3 C13H17ClN2O7S C21H28Cl2N2O9 C15H21ClN2O7S C14H18Cl2N2O3
QWBA labeled labeled labeled labeled unlabeled
347.09
348.07 348.93
349.09
350.09 351.09
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
347.09
348.10
349.09
350.09 351.09
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C15H20Cl2N2O3, M+nH ,347.090.0
0.5
1.0
1.5
8x10Intens.
0
1000
2000
3000
4000
5000
347 348 349 350 351 352 353 m/z
Contribution to QWBA abundance
Theoretical
Measured
Name Raclopride M1 M2 M3 M4 Measured m/z 347.0922 m/z 381.0519 m/z 523.1245 m/z 409.0814 m/z 333.0751Theoretical m/z 347.0924 m/z 381.0518 m/z 523.1246 m/z 409.0831 m/z 333.07672Formula C15H20Cl2N2O3 C13H17ClN2O7S C21H28Cl2N2O9 C15H21ClN2O7S C14H18Cl2N2O3
QWBA labeled labeled labeled labeled unlabeled
381.01
381.08
381.89
383.03
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
381.05
382.06
383.05
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C13H17ClN2O7S, M+nH ,381.050.0
0.2
0.4
0.6
0.8
7x10Intens.
0
1000
2000
3000
4000
5000
381.00 381.25 381.50 381.75 382.00 382.25 382.50 382.75 383.00 m/z
381.0111
381.0794
clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: +MS
381.0518clm glass liver manual 20120426 RAC 2mgkgn 5min s1_000001.d: C13H17ClN2O7S, M+nH ,381.05
0.0
0.2
0.4
0.6
0.8
7x10Intens.
0
1000
2000
3000
4000
5000
381.00 381.02 381.04 381.06 381.08 381.10 381.12 381.14 381.16 m/z
m/z 381.0111 m/z 381.0794
m/z 381.0518
Contribution to QWBA abundance
Theoretical
Measured
Theoretical isotope distribution
Measured isotope distribution
30 minute after doseRaclopride 2 mg/kg
Name Raclopride M1 M2 M3 M4 Measured m/z 347.0922 m/z 381.0519 m/z 523.1245 m/z 409.0814 m/z 333.0751Theoretical m/z 347.0924 m/z 381.0518 m/z 523.1246 m/z 409.0831 m/z 333.07672Formula C15H20Cl2N2O3 C13H17ClN2O7S C21H28Cl2N2O9 C15H21ClN2O7S C14H18Cl2N2O3
QWBA labeled labeled labeled labeled unlabeled
523.124
523.301 523.539
524.115
524.274
524.543
525.119
525.282
526.242
527.176
527.481
527.958
528.185
glass kidney 2mg_kg cortex_000001.d: +MS
523.124
524.128
525.122
526.125
527.119
528.122
glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.130
2
4
6
8
7x10Intens.
0
1000
2000
3000
4000
5000
523 524 525 526 527 528 m/z
Theoretical
Measured
Name Raclopride M1 M2 M3 M4 Measured m/z 347.0922 m/z 381.0519 m/z 523.1245 m/z 409.0814 m/z 333.0751Theoretical m/z 347.0924 m/z 381.0518 m/z 523.1246 m/z 409.0831 m/z 333.07672Formula C15H20Cl2N2O3 C13H17ClN2O7S C21H28Cl2N2O9 C15H21ClN2O7S C14H18Cl2N2O3
QWBA labeled labeled labeled labeled unlabeled
523.124
523.301 523.539
524.115
524.274
524.543
525.119
525.282
526.242
527.176
527.481
527.958
528.185
glass kidney 2mg_kg cortex_000001.d: +MS
523.124
524.128
525.122
526.125
527.119
528.122
glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.130
2
4
6
8
7x10Intens.
0
1000
2000
3000
4000
5000
523 524 525 526 527 528 m/z
523
glass kidney 2mg_kg cortex_000001.d: +MS
523glass kidney 2mg_kg cortex_000001.d: C21H28Cl2N2O9, M+nH ,523.13
0
1
2
3
4
7x10Intens.
0
1000
2000
3000
4000
5000
523.06 523.08 523.10 523.12 523.14 523.16 523.18 523.20 m/z
Theoretical isotope distribution
Measured isotope distribution
m/z 526.1246
m/z 526.1245
Theoretical
Measured
30 minute after doseRaclopride 2 mg/kg
Name Raclopride M1 M2 M3 M4 Measured m/z 347.0922 m/z 381.0519 m/z 523.1245 m/z 409.0814 m/z 333.0751Theoretical m/z 347.0924 m/z 381.0518 m/z 523.1246 m/z 409.0831 m/z 333.07672Formula C15H20Cl2N2O3 C13H17ClN2O7S C21H28Cl2N2O9 C15H21ClN2O7S C14H18Cl2N2O3
QWBA labeled labeled labeled labeled unlabeled
S.D 7%
S.D 56%N = 7
QWBA 30 minute after doseRaclopride 2 mg/kg
Dissected kidney 30 minute after dose raclopride 2 mg/kg
MSI m/z 347
Whole body
Tissue specific
Relative distribution
Absolute Quant.
Label available
MALDI MS imaging
ESI LESAprofiling
QWBA
Time scale&
Scope of analysis
Study
Summary
Fast
Cheap
Effective
AcknowledgmentsAcknowledgments
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 UniversityPer AndrenAnna Nilsson - Oral – Tue 2:50 (Ballroom B)
AstraZeneca AdvionSuzanne Iverson Mark Allen (UK)Goran Eklund Mark Baumert (UK)Daniel Borg
