A Practical 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; David H.G. Crout; Peter J. Derrick

doi:10.1255/ejms.385

journal article Feb 01, 2001

A Practical 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

David H.G. Crout Peter J. Derrick

European Journal of Mass Spectrometry Vol. 7 No. 1 pp. 29-34 · SAGE Publications

View at Publisher Save 10.1255/ejms.385

Abstract

Aquick, 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 heat treatment.

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Details

Published: Feb 01, 2001
Vol/Issue: 7(1)
Pages: 29-34
License: View

Authors

M

Michaela Scigelova