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. 2000 Feb;9(2):427–431. doi: 10.1110/ps.9.2.427

Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry.

J L Larson 1, E Ko 1, A D Miranker 1
PMCID: PMC2144543  PMID: 10716196

Abstract

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.

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Selected References

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  1. Chiti F., Webster P., Taddei N., Clark A., Stefani M., Ramponi G., Dobson C. M. Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3590–3594. doi: 10.1073/pnas.96.7.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cooper G. J., Willis A. C., Clark A., Turner R. C., Sim R. B., Reid K. B. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8628–8632. doi: 10.1073/pnas.84.23.8628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Esler W. P., Stimson E. R., Ghilardi J. R., Felix A. M., Lu Y. A., Vinters H. V., Mantyh P. W., Maggio J. E. A beta deposition inhibitor screen using synthetic amyloid. Nat Biotechnol. 1997 Mar;15(3):258–263. doi: 10.1038/nbt0397-258. [DOI] [PubMed] [Google Scholar]
  4. Goldsbury C., Kistler J., Aebi U., Arvinte T., Cooper G. J. Watching amyloid fibrils grow by time-lapse atomic force microscopy. J Mol Biol. 1999 Jan 8;285(1):33–39. doi: 10.1006/jmbi.1998.2299. [DOI] [PubMed] [Google Scholar]
  5. Guijarro J. I., Sunde M., Jones J. A., Campbell I. D., Dobson C. M. Amyloid fibril formation by an SH3 domain. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4224–4228. doi: 10.1073/pnas.95.8.4224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harper J. D., Lansbury P. T., Jr Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem. 1997;66:385–407. doi: 10.1146/annurev.biochem.66.1.385. [DOI] [PubMed] [Google Scholar]
  7. Kahn S. E., Andrikopoulos S., Verchere C. B. Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes. Diabetes. 1999 Feb;48(2):241–253. doi: 10.2337/diabetes.48.2.241. [DOI] [PubMed] [Google Scholar]
  8. Kayed R., Bernhagen J., Greenfield N., Sweimeh K., Brunner H., Voelter W., Kapurniotu A. Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vitro. J Mol Biol. 1999 Apr 9;287(4):781–796. doi: 10.1006/jmbi.1999.2646. [DOI] [PubMed] [Google Scholar]
  9. Kelly J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr Opin Struct Biol. 1998 Feb;8(1):101–106. doi: 10.1016/s0959-440x(98)80016-x. [DOI] [PubMed] [Google Scholar]
  10. Kudva Y. C., Mueske C., Butler P. C., Eberhardt N. L. A novel assay in vitro of human islet amyloid polypeptide amyloidogenesis and effects of insulin secretory vesicle peptides on amyloid formation. Biochem J. 1998 May 1;331(Pt 3):809–813. doi: 10.1042/bj3310809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. LeVine H., 3rd Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993 Mar;2(3):404–410. doi: 10.1002/pro.5560020312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lomakin A., Chung D. S., Benedek G. B., Kirschner D. A., Teplow D. B. On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1125–1129. doi: 10.1073/pnas.93.3.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loo J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev. 1997 Jan-Feb;16(1):1–23. doi: 10.1002/(SICI)1098-2787(1997)16:1<1::AID-MAS1>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  14. Lorenzo A., Razzaboni B., Weir G. C., Yankner B. A. Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus. Nature. 1994 Apr 21;368(6473):756–760. doi: 10.1038/368756a0. [DOI] [PubMed] [Google Scholar]
  15. Sunde M., Serpell L. C., Bartlam M., Fraser P. E., Pepys M. B., Blake C. C. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J Mol Biol. 1997 Oct 31;273(3):729–739. doi: 10.1006/jmbi.1997.1348. [DOI] [PubMed] [Google Scholar]
  16. Westermark P., Engström U., Johnson K. H., Westermark G. T., Betsholtz C. Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5036–5040. doi: 10.1073/pnas.87.13.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]

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