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Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS

  • Original Research
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International Journal for Ion Mobility Spectrometry

Abstract

Amyloid diseases are a serious cause for concern world-wide. To understand the mechanism of formation of the fibrillar structures associated with such disorders, it is necessary to study the progression from soluble protein or peptide monomer through an array of oligomers to the final, insoluble, fibrils. The protein IAPP is found in vivo in the form of insoluble amyloid deposits in the pancreatic islets of diabetes type II sufferers. Here, we have studied the in vitro self-aggregation of three fibril-forming peptides from the amyloidogenic core of IAPP. Using electrospray ionization—mass spectrometry coupled with ion mobility spectrometry, the mass and cross-sectional area of each oligomer present in the heterogeneous assembly mixtures can be determined individually in a single, rapid experiment over time. For the three peptides studied, oligomers ≤20-mer were characterized. Conversely, no oligomers higher than a dimer were detected for a non-assembling peptide control. The rate in which the cross-sectional area of the oligomers increases with increasing number of peptide sub-units indicates that assembly for the amyloid-forming peptides proceeds in a linear fashion until an oligomer of a certain size is attained. After this, a step increase in cross-sectional area occurs for the next higher-order oligomer. This behaviour can be explained by molecular modelling of singly, doubly, triply and quadruply stacked β-stranded structures. Using one peptide as an example, the cross-sectional areas of the lower order oligomers (dimer to pentamer) were found to be consistent with a single β-sheet model, whereas the higher order oligomers were consistent with double-stranded (hexamer to decamer oligomers), triply-stranded (11-mers to 15-mers) and quadruply-stranded (16-mers to 20-mers) β-sheet models.

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References

  1. Hoaglund CS, Valentine SJ, Sporleder CR, Reilly JP, Clemmer DE (1998) Three-dimensional ion mobility/TOFMS analysis of electrosprayed biomolecules. Anal Chem 70:2236–2242

    Article  CAS  Google Scholar 

  2. Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, Bateman RH, Bowers MT, Scrivens JH (2007) An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom 261:1–12

    Article  CAS  Google Scholar 

  3. Kemper PR, Dupuis NF, Bowers MT (2009) A new, higher resolution, ion mobility mass spectrometer. Int J Mass Spectrom 287:46–57

    Article  CAS  Google Scholar 

  4. Shepherd DA, Veesler D, Lichiere J, Ashcroft AE, Cambillau C (2011) Unraveling lactococcal phage baseplate assembly by mass spectrometry. Mol Cell Proteomics 10:M111.0009787

    Google Scholar 

  5. Uetrecht C, Versluis C, Watts NR, Wingfield PT, Steven AC, Heck AJ (2008) Stability and shape of hepatitis B virus capsids in vacuo. Angew Chem Int Ed Engl 47:6247–6251

    Article  CAS  Google Scholar 

  6. Ashcroft AE (2005) Recent developments in electrospray ionisation mass spectrometry: noncovalently bound protein complexes. Nat Prod Rep 22:452–464

    Article  CAS  Google Scholar 

  7. Zhou M, Morgner N, Barrera NP, Politis A, Isaacson SC, Matak-Vinkovic D, Murata T, Bernal RA, Stock D, Robinson CV (2011) Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334:380–385

    Article  CAS  Google Scholar 

  8. Benesch JL, Ruotolo BT, Simmons DA, Robinson CV (2007) Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem Rev 107:3544–3567

    Article  CAS  Google Scholar 

  9. Bich C, Zenobi R (2009) Mass spectrometry of large complexes. Curr Opin Chem Biol 19:632–639

    CAS  Google Scholar 

  10. Heck AJ (2008) Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods 11:927–933

    Article  Google Scholar 

  11. Loo JA (1997) Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev 16:1–23

    Article  CAS  Google Scholar 

  12. Smith DP, Radford SE, Ashcroft AE (2010) Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc Natl Acad Sci USA 107:6794–6798

    Article  CAS  Google Scholar 

  13. Bernstein SL, Dupuis NF, Laz ND, Wyttenbach T, Condron MM, Bitan G, Teplow BD, Shea J, Ruotolo BT, Robinson CV, Bowers MT (2009) Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nat Chem 1:326–331

    Article  CAS  Google Scholar 

  14. Kloniecki M, Jablonowska A, Poznanski J, Langridge J, Hughes C, Campuzano I, Giles K, Dadlez M (2011) Ion mobility separation coupled with MS detects two structural states of Alzheimer’s disease Aβ1-40 peptide oligomers. J Mol Biol 407:110–124

    Article  CAS  Google Scholar 

  15. Ashcroft AE (2010) Mass spectrometry and the amyloid problem—how far can we go in the gas phase? J Am Soc Mass Spectrom 21:1087–1096

    Article  CAS  Google Scholar 

  16. Sipe JD, Benson MD, Buxbaum JN, Ikeda S, Merlini G, Saraiva MJ, Westermark P (2010) Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the international society of amyloidosis. Amyloid 17:101–104

    Article  CAS  Google Scholar 

  17. Shewmaker F, McGlinchey RP, Wickner RB (2011) Structural insights into functional and pathological amyloid. J Biol Chem 286:16533–16540

    Article  CAS  Google Scholar 

  18. Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Methods 10:10–17

    Article  Google Scholar 

  19. Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Ann Rev Biochem 75:333–366

    Article  CAS  Google Scholar 

  20. Caughey B, Lansbury PT (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Ann Rev Neurosci 26:267–298

    Article  CAS  Google Scholar 

  21. Last NB, Rhoades E, Miranker AD (2011) Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity. Proc Natl Acad Sci USA 108:9460–9465

    Article  CAS  Google Scholar 

  22. Smith DP, Woods LA, Radford SE, Ashcroft AE (2011) Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly. Biophys J 101:1238–1247

    Article  CAS  Google Scholar 

  23. Bernstein SL, Liu D, Wyttenbach T, Bowers MT, Lee JC, Gray HB, Winkler JR (2004) Alpha-synuclein: stable compact and extended monomeric structures and pH dependence of dimer formation. J Am Soc Mass Spectrom 15:1435–1443

    Article  CAS  Google Scholar 

  24. Dupuis NF, Wu C, Shea J-E, Bowers MT (2009) Human islet amyloid polypeptide monomers form ordered β-hairpins: a possible direct amyloidogenic precursor. J Am Chem Soc 131:18283–18292

    Article  CAS  Google Scholar 

  25. Dupuis NF, Wu C, Shea JE, Bowers MT (2011) The amyloid formation mechanism in human IAPP: dimers have β-strand monomer—monomer interfaces. J Am Chem Soc 133:7240–7243

    Article  CAS  Google Scholar 

  26. Grabenauer M, Wu C, Soto P, Shea JE, Bowers MT (2009) Oligomers of the prion protein fragment 106–126 are likely assembled from β-hairpins in solution, and methionine oxidation inhibits assembly without altering the peptide’s monomeric conformation. J Am Chem Soc 132:532–539

    Google Scholar 

  27. Stridsberg M, Wilander E (1991) Islet amyloid polypeptide (IAPP). A short review. Acta Oncol 30:451–456

    Article  CAS  Google Scholar 

  28. Westermark P, Andersson A, Westermark GT (2011) Islet amyloid polypeptide, islet amyloid, and diabetes mellitus. Physiol Rev 91:795–826

    Article  CAS  Google Scholar 

  29. Westermark P, Engstrom U, Johnson KH, Westermark GT, Betsholtz C (1990) Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. Proc Natl Acad Sci USA 87:5036–5040

    Article  CAS  Google Scholar 

  30. Larson JL, Ko E, Miranker AD (2000) Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry. Prot Sci 9:427–431

    Article  CAS  Google Scholar 

  31. Zhao J, Yu X, Liang G, Zheng J (2011) Heterogeneous triangular structures of human islet amyloid polypeptide (amylin) with internal hydrophobic cavity and external wrapping morphology reveal the polymorphic nature of amyloid fibrils. Biomacromolecules 12:1781–1784

    Article  CAS  Google Scholar 

  32. Tenidis K, Waldner M, Bernhagen J, Fischle W, Bergmann M, Weber M, Merkle ML, Voelter W, Brunner H, Kapurniotu A (2000) Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties. J Mol Biol 295:1055–1071

    Article  CAS  Google Scholar 

  33. Moriarty DF, Raleigh DP (1999) Effects of sequential proline substitutions on amyloid formation by human amylin 20–29. Biochemistry 38:1811–1818

    Article  CAS  Google Scholar 

  34. Glenner GG, Eanes D, Wiley C (1988) Amyloid fibrils formed from a segment of the pancreatic islet amyloid protein. Biochem Biophys Res Commun 155:608–614

    Article  CAS  Google Scholar 

  35. Giles K, Pringle SD, Worthington KR, Little D, Wildgoose JL, Bateman RH (2004) Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun Mass Spectrom 18:2401–2414

    Article  CAS  Google Scholar 

  36. Smith DP, Knapman TW, Campuzano I, Malham RW, Berryman JT, Radford SE, Ashcroft AE (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry—mass spectrometry studies. Eur J Mass Spectrom 15:113–130

    Article  CAS  Google Scholar 

  37. Valentine SJ, Counterman AE, Clemmer DE (1999) A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections. J Am Soc Mass Spectrom 10:1188–1211

    Article  CAS  Google Scholar 

  38. Platt GW, Routledge KE, Homans SW, Radford SE (2008) Fibril growth kinetics reveal a region of beta 2-microglobulin important for nucleation and elongation of aggregation. J Mol Biol 378:251–263

    Article  CAS  Google Scholar 

  39. Madine J, Jack E, Stockley PG, Radford SE, Serpell LC, Middleton DA (2008) Structural insights into the polymorphism of amyloid-like fibrils formed by region 20–29 of amylin revealed by solid-state NMR and x-ray fiber diffraction. J Am Chem Soc 130:14990–15001

    Article  Google Scholar 

  40. Middleton DA (2011) Solid-state NMR detection of 14N-13C dipolar couplings between amino acid side groups provides constraints on amyloid fibril architecture. Magn Reson Chem 49:65–69

    Article  CAS  Google Scholar 

  41. Macke T, Case DA (1998) Modelling unusual nucleic acid structures. In: Leontes NB, SantaLucia J Jr (eds) Molecular modelling of nucleic acids. American Chemical Society, Washington, DC, pp 379–393

    Google Scholar 

  42. Case DA, Cheatham TE, Darden TA, Gohlke H, Luo R, Merz KM, Onufriev C, Simmerling C, Wang B, Woods RJ (2005) The amber biomolecular simulation programs. J Comput Chem 26:1668–1688

    Article  CAS  Google Scholar 

  43. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 36:1781–1802

    Article  Google Scholar 

  44. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple amber force fields and development of improved backbone parameters. Proteins 65:712–725

    Article  CAS  Google Scholar 

  45. Mesleh MF, Hunter JM, Shvartsburg AA, Schatz GC, Jarrold MF (1996) Structural information from ion mobility measurements: effects of the long-range potential. J Phys Chem 100:16082–16086

    Article  CAS  Google Scholar 

  46. Ashburn TT, Lansbury PT (1993) Interspecies variations affect the kinetics and thermodynamics of amyloid plaque formation; peptide models of pancreatic amyloid. J Am Chem Soc 115:11012–11013

    Article  CAS  Google Scholar 

  47. Wiltzius JJW, Sievers SA, Sawaya MR, Cascio D, Popov D, Riekel C, Eisenberg D (2008) Atomic structure of the cross-beta spine of uslet amyloid polypeptide (amylin). Prot Sci 17:1467–1474

    Article  CAS  Google Scholar 

  48. Ruotolo BT, Benesch JL, Sandercock AM, Hyung SJ, Robinson CV (2008) Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3:1139–1152

    Article  CAS  Google Scholar 

  49. Bleiholder C, Dupuis NF, Wyttenback T, Bowers MT (2011) Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation. Nat Chem 3:172–177

    Article  CAS  Google Scholar 

  50. Nielsen JT, Bjerring M, Jeppesen MD, Pedersen RO, Pedersen JM, Hein KL, Vosegaard T, Skrydstrup T, Otzen DE, Nielsen NC (2009) Unique identification of supramolecular structures in amyloid fibrils by solid-state NMR spectroscopy. Angew Chem Int Ed Engl 48:2118–2121

    Article  CAS  Google Scholar 

  51. Straaso LA, Nielsen NC (2010) Recoupling of native homonuclear dipolar couplings in magic-angle-spinning solid-state NMR by the double-oscillating field technique. J Chem Phys 133:064501

    Article  Google Scholar 

  52. Smith AM, Jahn TR, Ashcroft AE, Radford SE (2006) Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry. J Mol Biol 364:9–19

    Article  CAS  Google Scholar 

  53. World Health Organisation (2012) http://www.who.int/features/factfiles/diabetes. Accessed 6 Nov 2012

  54. Hoppener JWM, Nieuwenhuis MG, Vroom TM, Ahren B, Lips CJM (2002) Role of islet amyloid in type 2 diabetes mellitus: consequence or cause? Mol Cell Endocrinol 197:205–212

    Article  CAS  Google Scholar 

  55. Cao P, Raleigh DP (2012) Analysis of the inhibition and remodeling of islet amyloid polypeptide amyloid fibers by flavanols. Biochemistry 51:2670–2683

    Article  CAS  Google Scholar 

  56. Cheng B, Gong H, Li X, Sun Y, Zhang X, Chen H, Liu X, Zheng L, Huang K (2012) Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide. Biochem Biophys Res Commun 419:495–499

    Article  CAS  Google Scholar 

  57. Cheng B, Liu X, Gong H, Huang L, Chen H, Zhang X, Li C, Yang M, Ma B, Jiao L, Zheng L, Huang K (2011) Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus. J Agric Food Chem 59:13147–13155

    Article  CAS  Google Scholar 

  58. Trikha S, Jeremic AM, Zraika S, Hull RL, Verchere CB, Clark A, Potter KJ, Fraser PE, Raleigh DP, Khan SE (2011) Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol. J Biol Chem 286:36086–36097

    Article  CAS  Google Scholar 

  59. Zraika S, Hull RL, Verchere CB, Clark A, Potter KJ, Fraser PE, Raleigh DP, Kahn SE (2010) Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence? Diabetologia 53:1046–1056

    Article  CAS  Google Scholar 

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Acknowledgements

LY is funded by a Biotechnology and Biological Sciences Research Council CASE studentship (Grant Number BB/I015361/1) sponsored by Micromass UK Ltd/Waters Corpn, Manchester, UK; HN and TWK were funded by Engineering and Physical Sciences Research Council White Rose studentships (Grant numbers EP/G500010/1 and EP/E501869/1, respectively). The Synapt HDMS mass spectrometer was purchased with funds from the Biotechnology and Biological Sciences Research Council through its Research Equipment Initiative scheme (BB/E012558/1). We thank all members of the Ashcroft, Harris and Radford groups for helpful discussions.

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  1. Tom W. Knapman

    Present address: AB Sciex, Phoenix House, Lakeside Drive, Centre Park, Warrington, WA1 1RX, UK

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Lydia Young, Hlengisizwe Ndlovu, Tom W. Knapman, Sarah A. Harris, Sheena E. Radford & Alison E. Ashcroft

  2. School of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK

    Lydia Young, Sheena E. Radford & Alison E. Ashcroft

  3. School of Physics & Astronomy, Faculty of Maths & Physical Sciences, University of Leeds, Leeds, LS2 9JT, UK

    Hlengisizwe Ndlovu, Tom W. Knapman & Sarah A. Harris

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  1. Lydia Young
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Correspondence to Sheena E. Radford or Alison E. Ashcroft.

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Young, L., Ndlovu, H., Knapman, T.W. et al. Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS. Int. J. Ion Mobil. Spec. 16, 29–39 (2013). https://doi.org/10.1007/s12127-012-0115-z

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