Free energy for blue copper protein unfolding determined by electrospray ionisation mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determin ation of the free energy (AG) of unfolding of proteins is described. The me thod was tested using three blue copper proteins: wild type azurin, Cys-3Al a/Cys-26Ala (C3A/C26A) azurin mutant and wild-type amicyanin. The time cour se of the denaturation process of the proteins dissolved in methanol/water (50:50, v/v, pH 3.5) was followed by recording ESI mass spectra at time int ervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of the se two series of peaks at increasing time and at equilibrium, the free ener gy for the unfolding process for the three proteins could be determined. To evaluate the reliability of the thermodynamic data obtained by the ESI mas s spectrometric approach, the denaturation process was followed by UV-VIS s pectroscopy. The two sets of data obtained by these independent methods wer e in good agreement indicating that the ESI-MS approach can be used to obta in reliable quantitative information about the protein unfolding process. I n principle, this approach can be applied to other proteins and requires ve ry low amounts of sample, due to the intrinsic sensitivity of mass spectrom etry. This may prove particularly useful when the amount of sample availabl e prevents the use of current methods. Copyright (C) 2001 John Wiley & Sons , Ltd.