A pratical protocol for the reduction of disulfide bonds in proteins prior to analysis by mass spectrometry

Michaela Scigelova*, Philip S. Green, Anastassios E. Giannakopulos, Alison Rodger, D. H G Crout, Peter J. Derrick

*Corresponding author for this work

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

A quick, simple applicable protocol for the reduction of protein disulfide bonds for the purposes of mass spectrometry has been established. The method utilises the chemical reducing agent dithiothreitol. Proteins with various numbers of disulfide bonds per molecule were chosen for the study to demonstrate the general applicability of the method. The results obtained under controlled conditions (concentration of reagents, pH, temperature) showed that a five-minute treatment at 70°C with 10 mM dithiothreitol in 5 mM ammonium acetate buffer pH 5.5 was sufficient for complete reduction of disulfide bonds in all investigated proteins (α-lactalbumin, lysozyme, ribonuclease, oxytocin and wheat germ agglutinin). The progress of disulfide bond reduction was observed by electrospray ionisation and Fourier transform ion cyclotron resonance mass spectrometry. Circular dichroism was used to monitor conformational changes of reduced proteins and of their unreduced counterparts undergoing the same treatment.

Original languageEnglish
Pages (from-to)29-34
Number of pages6
JournalEuropean Journal of Mass Spectrometry
Volume7
Issue number1
Publication statusPublished - 2001
Externally publishedYes

Keywords

  • Circular dichroism
  • Disulfide bond
  • Electrospray ionisation
  • FTICR
  • Mass spectrometry
  • Protein

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    Scigelova, M., Green, P. S., Giannakopulos, A. E., Rodger, A., Crout, D. H. G., & Derrick, P. J. (2001). A pratical protocol for the reduction of disulfide bonds in proteins prior to analysis by mass spectrometry. European Journal of Mass Spectrometry, 7(1), 29-34.