Skip to main content

Quantitative Measurement of Fibrillogenesis by Mass Spectrometry

  • Protocol
Amyloid Proteins

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 299))

  • 3339 Accesses

Abstract

In this chapter, a method for the quantitative determination of amyloid conversion by electrospray mass spectrometry is presented. Mass spectrometry is typically used for the purpose of measuring the mass of unknowns. However, the judicious selection of an internal standard permits the quantitative determination of protein concentration. For amyloid formation, this is particularly useful in circumstances where either the protein under study is in limited abundance, or separation of the precursor from other protein factors is impractical or undesirable. For the measurement of amyloid formation, internal standards are typically mass distinct variants of the amyloid precursor. In addition, the extreme stability of amyloid fibers permits assessment of residual precursor concentration with or without separation of fibers from unreacted precursor. Lastly, by using internal standards which are not amyloidogenic and do not interfere with fiber formation, electrospray mass spectrometry permits quantitative measurement of fiber formation in real-time and with small (pmoles) quantities of protein.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 134.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kelly, J. W. (1998) The alternative conformations of amyloidogenic proteins and their multistep assembly pathways. Curr. Opin. Struct. Biol. 8, 101–106.

    Article  PubMed  CAS  Google Scholar 

  2. Rochet, J. C. and Lansbury, P. T. Jr. (2000) Amyloid fibrillogenesis: themes and variations. Curr. Opin. Struct. Biol. 10, 60–68.

    Article  PubMed  CAS  Google Scholar 

  3. Larson, J. L., Ko, E., and Miranker, A. D. (2000) Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry. Protein Sci. 9, 427–431.

    Article  PubMed  CAS  Google Scholar 

  4. Hoppener, J. W. M., Ahren, B., and Lips, C. J. M. (2000) Islet amyloid and type 2 diabetes mellitus. N. Engl. J. Med. 343, 411–419.

    Article  PubMed  CAS  Google Scholar 

  5. Jaikaran, E. T. and Clark, A. (2001) Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology. Biochim. Biophys. Acta 1537, 179–203.

    PubMed  CAS  Google Scholar 

  6. Hooke, S. D., Eyles, S. J., Miranker, A., Radford, S. E., Robinson, C. V., and Dobson, C. M. (1995) Cooperative elements in protein-folding monitored by electrosprayionization mass-spectrometry. J. Am. Chem. Soc. 117, 7548–7549.

    Article  CAS  Google Scholar 

  7. Canet, D., Last, A. M., Tito, P., et al. (2002) Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme. Nat. Struct. Biol. 9, 308–315.

    Article  PubMed  CAS  Google Scholar 

  8. Eakin, C. M., Knight, J. D., Morgan, C. J., Gelfand, M. A., and Miranker, A. D. (2002) Formation of a copper specific binding site in non-native states of beta-2-microglobulin. Biochemistry 41, 10646–10656.

    Article  PubMed  CAS  Google Scholar 

  9. Oda, Y., Huang, K., Cross, F. R., Cowburn, D., and Chait, B. T. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596.

    Article  PubMed  CAS  Google Scholar 

  10. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.

    Article  PubMed  CAS  Google Scholar 

  11. LeVine, H. D. (1993) Thioflavine T interaction with synthetic Alzheimer%s disease betaamyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404–410.

    Article  PubMed  CAS  Google Scholar 

  12. Padrick, S. B. and Miranker, A. D. (2002) Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis. Biochemistry 41, 4694–4703.

    Article  PubMed  CAS  Google Scholar 

  13. Kebarle, P. (2000) A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J. Mass Spectrom 35, 804–817.

    Article  PubMed  CAS  Google Scholar 

  14. Cole, R. B. (2000) Some tenets pertaining to electrospray ionization mass spectrometry. J. Mass Spectrom 35, 763–772.

    Article  PubMed  CAS  Google Scholar 

  15. Westermark, P., Engstrom, U., Johnson, K. H., Westermark, G. T., and Betsholtz, C. (1990) Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. Proc. Natl. Acad. Sci. USA 87, 5036–5040.

    Article  PubMed  CAS  Google Scholar 

  16. Larson, J. L. and Miranker, A. D. (2004) The mechanism of insulin action on islet amyloid polypeptide fiber formation. J. Mol. Biol. 35, 221–231.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Miranker, A.D. (2005). Quantitative Measurement of Fibrillogenesis by Mass Spectrometry. In: Sigurdsson, E.M. (eds) Amyloid Proteins. Methods in Molecular Biology™, vol 299. Humana Press. https://doi.org/10.1385/1-59259-874-9:185

Download citation

  • DOI: https://doi.org/10.1385/1-59259-874-9:185

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-337-4

  • Online ISBN: 978-1-59259-874-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics