Characterisation of cytochrome c unfolding by nano-electrospray-ionization time-of-fight and Fourier-transform-ion-cyclotron-resonance mass spectrometry

Eur. J. Mass Spectrom. 11, 519–524 (2005)
DOI: 10.1255/ejms.730

Characterisation of cytochrome c unfolding by nano-electrospray-ionization time-of-fight and Fourier-transform-ion-cyclotron-resonance mass spectrometry

Nikolay Youhnovski,^a Irena Matečko,^b Maria Šamalikova^b and Rita Grandori^b,*
^aDepartment of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany
^bInstitute of Organic Chemistry, Johannes Kepler University, Altenbergerstraße 69, 4040 Linz, Austria

ABSTRACT:

Protein charge-state distributions (CSDs) in electrospray-ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%-5% is found to favor cyt c folding at pH ~7. Furthermore, we perform comparison of CSDs obtained by time-of-flight (ToF) and Fourier-transform-ion- cyclotron-resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kind of denaturants. In particular, experiments with 1-propanol suggest that also by FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.

Keywords: Protein folding, folding intermediates, hydrophobic effect, electrostatic effect, secondary and tertiary protein structure

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