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

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 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. Ab(39-42) Modulates AbOligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011) Presentation by: Mahati Mokkarala Date of Presentation: 12/4/12 Protein misfolding characterization by ion mobility-mass spectrometry (IM-MS)

2. Mass Spectrometry- What is it? • Effective method for determining compound chemical structure, protein modification patterns, interactions, etc. Mass Spectrometer machine- mass analyzer/detector. Example: Time of Flight Mass Spectrometer (TOF), Quadrupole Mass Spectrometer (QMS) -Detects m/zz/n of various ion fragments Ionization- Hard or Soft methods (conversion to gaseous state) Ex: for proteins, ESI (nano), MALDI with lasers Protein Sample/ (can be liquid, other states)

3. 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]

4. 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])

5. 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].

6. 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

7. 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]

8. 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

9. 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)

10. 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.

11. Protocol- Paper 1 • 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 IM (from source [3] ESI/Quadrople Mass Spec. Image from: http://chemwiki.ucdavis.edu

12. 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:

13. 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

14. 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

15. Results-Paper 1 Can clearly correlate experimental collision cross section per each oligomer combination with calculated theoretical s(n) Top: for YGGFL Second: for NNQQNY

16. Results- Paper 1 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

17. Results- Paper 1 Physical Verification Verification of fibril formation at specified oligomer (n) verified by AFM visualization of each protein mixture sample

18. 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-bfibril transformation occurs • Very relevant for greater study of amyloidbcaused diseases

19. Paper 2 Problem/Results/Discussion Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. Ab(39-42) Modulates AbOligomerization but not Fibril Formation. Biochemistry. 51, 108-177 (2011)

20. Paper 2 – Problem/Question of Interest • Mechanism of Ab(39-42) binding to Ab42 or Ab40 tough to experimentally verify via X ray crystollagraphy or NMR • IM-MS and molecular dynamic simulations as well as ThT assays- further verify Ab(39-42) interactions with Ab42 and Ab40 • Why important?: CTF Ab(39-42) known to inhibit Abtoxicity

21. Paper 2- Methods/Techniques • IM- (nanospray) ESI- quadroplemass spectra- oligomer disassociation due to Ab(39-42) (CTF) • ThT fluorescence assay- does Ab(39-42)influence/limit fibril formation? • Modeling software- AMBER force field simulation, SHAKE- verify possible binding/structure Ab(39-42) with Abpeptide

22. 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 (Ab42)

23. 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 Ab42 particle – Does CTF reverse Abaggregation? Ans: Yes Prevention of dodecamers, decamers requires high (1:5) concentration CTF Incubation of select amyloiddimer peaks for 2 hours prior to exposure to CTF

24. Paper 2- cont. IM-MS results Question: Does CTF bind to tetramers, hexamers, dimers of amyloidb42? Ans: Yes Expose m/z = 1884 peak with bound CTF to dimers to IM-MS indicate definite cross sections for dimer, tetramer, hexamer

25. Paper 2- IM-MS for Ab40- CTF Interactions? IM-MS indicates- no shift oligomer size with CTF, same as peptide CTF- interacts with Ab40 Similar experiment repeated for the Ab40 peptide- as above, observe distinctive peaks z/n = -4, -3 for one or two CTF- binding to single oligomer One dimer-CTF species identified

26. Paper 2: Results, ThT and Toxicity Studies Both CTF plus Ab40/Ab42 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. Paper 2 Results: Modeling • Observe structure if CTF binds to Abmore 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 Ab42 peptide.

28. Paper 2 Conclusions: With IMS-MS techniques:. • CTF binds (Van der Waals) with monomeric, 2,4,6 Amyloidb 42 particles • CTF disassociates dodecamers into non toxic oligomers • Ab40 – binds with two CTF via electrostatic interaction, no disaggregation oligomers • CTF binding- C, N terminus, internal structures Amyloidb 42

29. 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. Ab(39-42) Modulates AbOligomerization 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)

30. Questions?