Gas Phase Interactions and Aggregation of Amyloid β -Proteins Using Ion Mobility - Bernstein et al....

7/30/2019 Gas Phase Interactions and Aggregation of Amyloid -Proteins Using Ion Mobility - Bernstein et al. - Unknown - Unknown

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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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Gas Phase Interactions and Aggregation of Amyloid β -Proteins Using Ion Mobility - Bernstein et al....

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