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

A quick, simple applicable protocol for the reduction of protein disulfide bonds for the purposes of mass spectrometry has been established. The metho d utilises the chemical reducing agent dithiothreitol. Proteins with variou s numbers of disulfide bonds per molecule were chosen for the study to demo nstrate 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 degreesC 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 (alpha -lactalbumin, lysozyme, ribonuclease, oxytocin and wheat germ agglutinin). The progress of disulfi de bond reduction was observed by electrospray ionisation and Fourier trans form ion cyclotron resonance mass spectrometry. Circular dichroism was used to monitor conformational changes of reduced proteins and of their unreduc ed counterparts undergoing the same heat treatment.