1. Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to  - 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  Modulates A  Oligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011) Presentation by: Mahati Mokkarala Date of Presentation: 12/4/12 1

2.  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 2

3.  Ion mobility devices separate (peptide sequence) ions based on particle mobility, shape, charge  Easily pair ion mobility with mass spectra and ionization devices [5] 3

4. 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]) 4

5.  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]. 5

6.  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 6

7.  (K naught) Reduced mobility ~ 1/  (  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] 7

8.  Bowers et al. Paper 2, summarizes key relationship between  collision cross- section) and drift time  q = ion charge, T = temperature,  reduced mass, N = He/gas number density, l = drift cell length 8

9. Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to  - sheet in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010) 9

10.  Detection oligomer shifts- tough to characterize due to quick conformational shifts  With IM-MS, could greater determine at oligomer combination (n) globular-  sheet transformation occurs. 10

11. 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 11

12.  IM (time of delay) – calculate  for each oligomer (size n)  Compare collision cross section per oligomer number (n) with theoretical  n) for fibril/isotropic growth Isotropic Growth formula: Fibril Growth formula: 12

13. Mass Spectra- indicates oligomerization due to large n/z observed for two peptides- YGGFL, VEALYL (one isotropic growth control, other fibril 13

14. 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 14

15. Can clearly correlate experimental collision cross section per each oligomer combination with calculated theoretical  n) Top: for YGGFL Second: for NNQQNY 15

16. Experimental 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 16

17. Verification of fibril formation at specified oligomer (n) verified by AFM visualization of each protein mixture sample 17

18.  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-  fibril transformation occurs  Very relevant for greater study of amyloid  caused diseases 18

19. Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. A  Modulates A  Oligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011) 19

20.  Mechanism of  binding to  or  tough to experimentally verify via X ray crystollagraphy or NMR  IM-MS and molecular dynamic simulations as well as ThT assays- further verify  interactions with  and   Why important?  CTF A  known to inhibit A  toxicity 20

21.  IM- (nanospray) ESI- quadrople mass spectra- oligomer disassociation due to  CTF)  ThT fluorescence assay- does   influence/limit fibril formation?  Modeling software- AMBER force field simulation, SHAKE- verify possible binding/structure  with  peptide 21

22. 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  22

23. n/z = -5/2 m/z = ~ 1800 for dimer peak- does CTF prevent dodecamers? Ans: Yes. n/z = -5/2 for A  particle – Does CTF reverse A  aggregation? 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 23

24. 24 Question: Does CTF bind to tetramers, hexamers, dimers of amyloid  Ans: Yes Expose m/z = 1884 peak with bound CTF to dimers to IM- MS indicate definite cross sections for dimer, tetramer, hexamer

25. 25 Similar experiment repeated for the A  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 

26. 26 Both CTF plus A  peptides 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

27. 27 Observe structure if CTF binds to A  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  peptide.

28. With IMS-MS techniques:.  CTF binds (Van der Waals) with monomeric, 2,4,6 Amyloid  42 particles  CTF disassociates dodecamers into non toxic oligomers   – binds with two CTF via electrostatic interaction, no disaggregation oligomers  CTF binding- C, N terminus, internal structures Amyloid  42 28

29.  [1] Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to  - sheet in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010)  [2] “Theories and Analysis.” The Bower’s Group UC Santa Barbara.. Accessed: December 3, 2012.http://bowers.chem.ucsb.edu/theory_analysis/  [3] Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. A  Modulates A  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) 29

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