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Protein misfolding characterization by ion mobility-mass spectrometry (IM-MS)

Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to b- sheet in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010)

Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. A (39-42) b Modulates A b Oligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011)

Presentation by: Mahati MokkaralaDate of Presentation: 12/4/12

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Mass Spectrometry- What is it?

Effective method for determining compound chemical structure, protein modification patterns, interactions, etc.

Protein Sample/ (can be liquid, other states)

Ionization- Hard or Soft methods (conversion to gaseous state) Ex: for proteins, ESI (nano), MALDI with lasers

Mass Spectrometer machine- mass analyzer/detector. Example: Time of Flight Mass Spectrometer (TOF), Quadrupole Mass Spectrometer (QMS)-Detects m/z z/n of various ion fragments

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Ion mobility-mass spectrometry (IM-MS)

Ion mobility devices separate (peptide sequence) ions based on particle mobility, shape, charge

Easily pair ion mobility with mass spectra and ionization devices [5]

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Physics of ion mobility in IM-MS

• Peptide ion fragments enter chamber filled with gas (buffer gas, chiral selectivity element, etc)• Ion mobility delayed- ‘friction’ collisions with gas molecules- propelled by electric field (Image from source [2])

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IM-MS Classification

Linear Drift Time (LDT) Mass Spectrometry- ‘easier’ calculation correlation between collision cross section and drift time for ions

Traveling Wave Ion Guide (TWIG) IM-MS

Field Asymmetric Ion Mobility Mass Spectrometry (FAIM) [4].

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Linear Drift Time Mass Spectrometry (LDT- Mass Spec.)

LDT gas tube- with weak electric field- constant drift velocity

Can average collisions to get the collision cross section

Advantages: high resolution, easier to quantify degree of ion separation

Disadvantages: low ‘drift cycle’ need to constantly introduce a pulse of ions- can promote wasting of a large portion of sample source [4]

Image from source [4] (see works cited), in source reprinted by permission from source [20] in paper

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LDT IM-MS Key Equations

(K naught) Reduced mobility ~ 1/ W( = W Collision Cross section Image from source [4]

How to calculate K naught? K simplified, related to drift time P- pressure, V- voltage, linear relationship Image from source [2]

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LDT IM-MS Key Equations cont.

Bowers et al. Paper 2, summarizes key relationship between (s collision cross-section) and drift time

q = ion charge, T = temperature, m= reduced mass, N = He/gas number density, l = drift cell length

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Paper 1 Problem/Results/Discussion

Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to b- sheet in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010)

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Problem/Question of Interest

Detection oligomer shifts- tough to characterize due to quick conformational shifts

With IM-MS, could greater determine at oligomer combination (n) globular-b sheet transformation occurs.

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Protocol- Paper 1

ESI/Quadrople Mass Spec. Image from: http://chemwiki.ucdavis.edu

IM (from source [3]

•amyloid-forming yeast prion protein Sup35 (NNQQNY)•human insulin regions- (VEALYL) •human islet amyloid polypeptide- (SSTNVG)•YGGFL- usually forms an exclusively isotropic not fibril structure

Peptides exposed to following apparatus:

And then

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Cont. Protocol Paper 1

IM (time of delay) – calculate s for each oligomer (size n)

Compare collision cross section per oligomer number (n) with theoretical (s n) for fibril/isotropic growth

Isotropic Growth formula: Fibril Growth formula:

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Results- Paper 1 –ESI- Quadrupole Mass Spectra

Mass Spectra- indicates oligomerization due to large n/z observed for two peptides- YGGFL, VEALYL (one isotropic growth control, other fibril

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Results- Paper 1- Supplementary Figures- Example of IM-MS ATD

Shows sample ATD intensity captures by IM-MS for the NNQQNY peptide; broad peaks- correlate to multiple oligomer combination states- use average drift time for calculations

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Results-Paper 1

Can clearly correlate experimental collision cross section per each oligomer combination with calculated theoretical (s n)

Top: for YGGFLSecond: for NNQQNY

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Results- Paper 1Experimental data and proposed oligomerization for peptide VEALYL (c )and peptide SSTNVG (d )

Indicates peptide (c ) –initiates with single strand fibril before at n =5 switching to the zipper form

Peptide (d)- isotropic until n = 12/14, consists of both zipper and isotropic form

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Results- Paper 1 Physical Verification

Verification of fibril formation at specified oligomer (n) verified by AFM visualization of each protein mixture sample

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Conclusions- Paper 1

With IMS-MS, now cab follow through peptide self-assembly step by step from an oligomer of 1 for given peptide fragment

Stresses importance of the IMS-MS technique can learn more on at what state oligomer- b fibril transformation occurs

Very relevant for greater study of amyloid b caused diseases

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Paper 2 Problem/Results/Discussion

Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. A (39-42) b Modulates A bOligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011)

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Paper 2 – Problem/Question of Interest

Mechanism of (39-42) Ab binding to 42Ab or 40 Ab tough to experimentally

verify via X ray crystollagraphy or NMR IM-MS and molecular dynamic

simulations as well as ThT assays- further verify (39-42) Ab interactions with 42 Ab and 40 Ab

Why important?: CTF A (39-42) bknown to inhibit A b toxicity

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Paper 2- Methods/Techniques IM- (nanospray) ESI- quadrople

mass spectra- oligomer disassociation due to (39-42) (Ab CTF)

ThT fluorescence assay- does (39-Ab42)influence/limit fibril formation?

Modeling software- AMBER force field simulation, SHAKE- verify possible binding/structure (39-42) Ab with Abpeptide

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Paper 2- ESI-Mass Spec. Results

Results- Definite difference in mass spectra peaks between both spectra a- Amyloid particle alone, b- Amyloid plus 1:5 CTF added

-Key peaks to focus on in b figure: z/n = -5/2 peak – one CTF/dimer

z/n = -3, 1 or 2 CTF bound to single oligomer (A 42) b

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Paper 2 – IM- MS results n/z = -5/2 m/z = ~ 1800 for dimer peak- does CTF prevent dodecamers? Ans: Yes.

n/z = -5/2 for A 42 bparticle – Does CTF reverse A baggregation? Ans: Yes

Incubation of select amyloid dimer peaks for 2 hours prior to exposure to CTF

Prevention of dodecamers, decamers requires high (1:5) concentration CTF

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Paper 2- cont. IM-MS results

Question: Does CTF bind to tetramers, hexamers, dimers of amyloid 42? bAns: Yes

Expose m/z = 1884 peak with bound CTF to dimers to IM-MS indicate definite cross sections for dimer, tetramer, hexamer

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Paper 2- IM-MS for 40- Ab CTF Interactions?

Similar experiment repeated for the A 40 b peptide- as above, observe distinctive peaks z/n = -4, -3 for one or two CTF- binding to single oligomer

One dimer-CTF species identified

IM-MS indicates- no shift oligomer size with CTF, same as peptide

CTF- interacts with A 40b

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Paper 2: Results, ThT and Toxicity Studies

Both CTF plus A 40/ 42 b Abpeptides with MTT assay-PC12 cells promotes cell viability –importance of breaking toxic oligomer aggregates

ThT fluorescence- EM microscope visualization-Fluorescence increase-fibrils--Oligomers eventually to fibrils even with CTF

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Paper 2 Results: Modeling

Observe structure if CTF binds to Ab more than 20 times, adds to being in a bound state, etc

Calculate cross sections of structures (long collision integral)

Compare structures to Mass Spec experimental data

Observe: CTF fragments bind: N, C terminus, internal regions via van der Walls interactions of 42 Ab peptide. 

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Paper 2 Conclusions:

With IMS-MS techniques:. CTF binds (Van der Waals) with monomeric, 2,4,6 Amyloid b

42 particles CTF disassociates dodecamers into non toxic oligomers 40Ab – binds with two CTF via electrostatic interaction, no

disaggregation oligomers CTF binding- C, N terminus, internal structures Amyloid b 42

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Works Cited

[1] Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to b- sheet in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010)

[2] “Theories and Analysis.” The Bower’s Group UC Santa Barbara. <http://bowers.chem.ucsb.edu/theory_analysis/> . Accessed: December 3, 2012.

[3] Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. A (39-42) b Modulates A b Oligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011)

[4] Harvey S.R. MacPhee C.E., Barran P.E. Ion mobility mass spectrometry for peptide analysis. Methods. 54(4), 454-461 (2011)

[5] Kanu A.B., Dwivedi P., Tam M., Matz L., Hill H.H., Ion mobility- mass spectrometry. Journal of Mass Spectrometry. 43, 1-22 (2008)

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Questions?