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Gas Phase Interactions and Aggregation
of Amyloid -ProteinsUsing Ion Mobility
Summer Bernstein, Thomas Wyttenbach, Gal Bitan,David Teplow, Michael T. Bowers
Bringham and Womans Hospital,
Harvard Institutes of Medicine
Department of Chemistry and Biochemistry
University of California, Santa Barbara
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Normal Brain Alzheimers Brain
www.alzheimers.org/pet.html
PET
BrainScan
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The Hallmarks of Alzheimers Disease
Neurofibrillary Tangles and SenilePlaques
Normal Alzheimers Disease
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Lumen
Cytoplasm
http://www.biocarta.com/pathfiles/appPathway.asp
APP
Amyloid plaques caused by
mutations on APP(Amyloid Protein Precursor,
Chromosome 21)
Beta and gamma secretase
enzymes can cut APP yielding
a harmless 40 and a toxic 42 (amyloid-peptide)
amino acid long chain
-Secretase
-Secretase
Formation of
Amyloid Plaques
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Comparison of A40 and A42 Small primary structural difference
Display distinct biological and clinical behavior
90% is A40 in soluble form
aggregate form in plaques is predominantly A42
A42 displays enhanced neurotoxicity
Ile-41: critical residue promoting oligomerization
Ala-42: facilitate self-association
Asp-
Ala-
Glu-
Phe-Arg-
His-
Asp-
Ser-Gly-
Tyr-
Glu-
Val-
His-
His-
Gln-
Lys-
Leu-Val
Phe-
Phe-
Ala-
Glu-
Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val -(Ile-Ala)
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Morphology of A AssembliesKirkitadze,M.D. J.Mol.Biol. (2001) 312,1103-1119
A40 and A42 form fibrils via different mechanisms
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Monomer (U) Paranuclei (U) Large oligomers (U)
(U//)
Protofibrils ()
5 nm
Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Te low D.B. 2002 PNAS earl addition.
A Simple Model of A42 Assembly
10nm
Fibrils ()
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Ion Funnel
Drift
Cell
Ion Optics
Quad
Analyzer
Detector
ESI
Source
To PumpTo PumpTo Pump
To Pump
Ion mobility-mass spectrometry setup
Ion
Source
IonSource
Drift
Cell
DriftCell MS
MS DetectorDetectorIon
Funnel
IonFunnel
in out
Drift cellE
15 torr He
Drift
Cell
DriftCell
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Ion arrival time
largecross
section
smallcross
section
Ion mobility spectrum:
Ion arrival time distribution (ATD)
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Mobilities (K) Collisioncross-sections()
Exp : ATDs
Time
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Ion Mobility as a Tool
Compare mass spectra of A40 and A42
Use IM to detect conformational differences and
aggregate formation in A40 and A42
Conduct kinetic studies of aggregate dissociation
Observe soluble intermediates
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6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0
6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0
+3
+4
+5Dimer
+7
+3
+4+5
Dimer+7+6
A42
A40
m/z
A40 and A42 Mass Specs
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600 8 00 100 0 1 200 140 0 1 600
52 VA2
7+
Mass Spectra Injection EnergyDependence for A40
m/z
600 80 0 1 00 0 12 00 140 0 16 00
100 V +3
+4
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+6
6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0
Intensity
m /z
+3
+4
+5
Dimer+7
Mass Spectrum of A(140)
Temperature(30V)
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0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
300K
340K
495K
540K
57 V
109 V
2 V
83 V
A(1-40)3+Injection EnergyTemperature(30V)
Time (ms)
Bradykinin M M A(1-40)3+
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30 V
60 V
75 V
105 V
1 V
Bradykinin
0.4 0.5 0.6 0.7 0.8
0.4 0.5 0.6 0.7 0.8
0.4 0.5 0.6 0.7 0.8
0.4 0.5 0.6 0.7 0.8
0.4 0.5 0.6 0.7 0.8
T D
M
T
D
M
DM
D
M
M
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
0.6 0.7 0.8 0.9
57 V
109 V
2 V
83 V
30 V
D
X
D X
D
XM
X
M A(1-40)
Time ms
A(1-40)
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0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8
0.5 0.6 0.7 0.8 0.9
M
X
XDf
MX
Df
300 K
450K
Monomer +3
time ms
Dimer +7
0.5 0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8
0.5 0.6 0.7 0.8
DsDf
time (ms)
A(1-40)
500K
X = Dimer?
Cross Section vs Charge State
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5 6 7 8
1050
1150
1250
1350
1450
1550
525
575
625
675
725
775
Charge State
Monomer Dimer
(
2) (
2)
D
M
X
A40
A42
Cross Section vs Charge StateA40 and A42
A(1 40)3+
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0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8 0.9
0.5 0.6 0.7 0.8
0.6 0.7 0.8 0.9
300K
340K
495K
540K
A(1-40)3+
Temperature(30V)
Time (ms)
D X
M X
D
M
M
X
D
X
M
320 K
510 K
?
A40
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5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
0.0017 0.0018 0.0019 0.002 0.0021 0.0022
1/T (K-1
)
ln(k)
A42
Ea=18.4 kcal/mol
A = 1.77 *1011s-1
S = -9.47 cal mol-1 K-1
A40
Ea= 28.9 kcal/mol
A = 6.7*1016 s-1
S
= 15.8 cal mol
-1
K
-1
X M
Arrhenius Analysis
A40
A42
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Mobilities (K) Collisioncross-sections()
Exp : ATDs
Th: Molecular
Dynamics
Candidate
Structures
Collision
cross-sections()
A40 Exp
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500
550
600
650
700
750
800
850
900
950
1000
-8 -6 -4 -2 0 2 4 6 8
A Monomers A40 CalcA42 Exp
A42 Calc
Charge State
CrossSectio
n(2)
-helix
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BrookhavenPDB
8002Calculated
(AMBER)
657 2
A(1-40)3-
Cross Section vs Charge State
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1050
1150
1250
1350
1450
1550
Charge State
Monomer Dimer
(
2) (
2)
D
M
X
525
575
625
675
725
775
A40 Exp
A40 Calc
A42 Exp
A42 Calc
Cross Section vs Charge StateA40 and A42
D
M
X
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A(40)26+2A(40)3+
Exp 6192
Calculated 6582
Exp 1004 2
Calculated 1046 2
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Summary and Future Work
Use Ion Mobility to obtain structural andkinetic information about A gas phaseaggregation
Exam Under Native Condition
Use IM to study other proteins prone for
aggregation (i.e. Prions)
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Acknowledgements
David Teplow Gal Bitan
Thomas Wyttenbach
Catie Carpenter
Bringham and Womans Hospital,Harvard Institutes of Medicine
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Bowers Group
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