Advertisement

Evaluating Amyloid Beta (Aβ) 1-40 Degradation by Capillary Electrophoresis

  • Benjamin J. Alper
  • Walter K. Schmidt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 984)

Abstract

Here, we describe a capillary electrophoresis method for evaluating proteolysis of the amyloid beta peptide (Aβ) 1–40. This method is suitable for kinetic studies, demands little specialized equipment, and consumes only small quantities of a commercially available substrate whose physiological accumulation is thought to underlie the development of Alzheimer’s disease.

Key words

Capillary electrophoresis Amyloid beta (Aβ) 1–40 Proteolysis Insulin-degrading enzyme 

Notes

Acknowledgment

We are grateful to Drs. Nandu Menon, Alan Przybyla, and Zachary Wood (all of the University of Georgia, Department of Biochemistry and Molecular Biology) for advice and assistance, and for access to experimental equipment and materials. Aβ 1–40 consumed during method development and analysis was a gift of rPeptide, Inc. (Athens, GA, USA).

References

  1. 1.
    Glenner GG, Wong CW (1984) Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Comm 120:885–890PubMedCrossRefGoogle Scholar
  2. 2.
    Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356PubMedCrossRefGoogle Scholar
  3. 3.
    McDermott JR, Gibson AM (1997) Degradation of Alzheimer’s beta-amyloid protein in human and rat brain peptidases: involvement of insulin-degrading enzyme. Neurochem Res 22:49–56PubMedCrossRefGoogle Scholar
  4. 4.
    Vekrellis K, Ye Z, Qiu WQ, Walsh D, Hartley D, Chesneau V et al (2000) Neurons regulate extracellular levels of amyloid-beta protein via proteolysis by insulin-degrading enzyme. J Neurosci 20:1657–1665PubMedGoogle Scholar
  5. 5.
    Mukherjee A, Song E, Kihiko-Ehmann M, Goodman JP, St Pyrek J, Estus S et al (2000) Insulysin hydrolyzes amyloid-beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques. J Neurosci 20:8745–8749PubMedGoogle Scholar
  6. 6.
    Miller BC, Eckman EA, Sambamurti K, Dobbs N, Chow KM, Eckman CB et al (2003) Amyloid beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo. Proc Natl Acad Sci USA 100:6221–6226PubMedCrossRefGoogle Scholar
  7. 7.
    Leissring MA, Farris W, Chang AY, Walsh DM, Wu X, Sun X et al (2003) Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40:1087–1093PubMedCrossRefGoogle Scholar
  8. 8.
    Varesio E, Rudaz S, Krause K-H, Veuthey J-L (2002) Nanoscale liquid chromatography and capillary electrophoresis coupled to electrospray mass spectrometry for the detection of amyloid beta peptide related to Alzheimer’s disease. J Chr A 974:135–142CrossRefGoogle Scholar
  9. 9.
    Sabella S, Quaglia M, Lanni C, Racchi M, Govoni S, Caccialanza G et al (2004) Capillary electrophoresis studies on the aggregation process of beta-amyloid 1–42 and 1–40 peptides. Electrophoresis 25:3186–3194PubMedCrossRefGoogle Scholar
  10. 10.
    Verpillot R, Otto M, Klafki H, Taverna M (2008) Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer’s disease. J Chr A 1214:157–164CrossRefGoogle Scholar
  11. 11.
    Alper BJ, Schmidt WK (2009) A capillary electrophoresis method for evaluation of Aβ proteolysis in vitro. J Neurosci Methods 178:40–45PubMedCrossRefGoogle Scholar
  12. 12.
    Gomori G (1955) Preparation of buffers for use in enzyme studies. Methods Enzymol 1:138–146CrossRefGoogle Scholar
  13. 13.
    Alper BJ, Rowse JW, Schmidt WK (2009) Yeast Ste23p shares functional similarities with mammalian insulin-degrading enzymes. Yeast 11:595–610CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of BiochemistrySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of GeorgiaAthensUSA

Personalised recommendations