Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

Authors

Stephen James Watt, University of Wollongong
Aaron J. Oakley, Australian National UniversityFollow
Margaret Sheil, University of WollongongFollow
Jennifer L. Beck, University of WollongongFollow

RIS ID

13159

Publication Details

Watt, S. James., Oakley, A. J., Sheil, M. & Beck, J. L. (2005). Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine. Rapid Communications in Mass Spectrometry, 19 (15), 2123-2130.

Abstract

The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca₄CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca₄CaM and Ca₄CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca₄CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16− ion was the most abundant ion observed for the apo form and the 8− ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca₄CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca₄CaM with TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca₄CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca₄CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.