Tandem mass spectrometry of protein-protein complexes: cytochrome c-cytochrome b5.
An improved method to interpret triple quadrupole MS/MS experiments of complexes of large ions is presented and applied to a study of the complex formed by the proteins cytochrome c and cytochrome b5. Modeling of the activation and dissociation process shows that most of the reaction occurs near the collision cell exit where ions have the highest internal energies. Experiments at different collision cell pressures or with different collision gases (Ne, Ar, Kr) are interpreted with a previously proposed collision model (Chen et al., Rapid Commun. Mass Spectrom. 1998, 12, 1003-1010) to calculate the internal energy added to ions to cause dissociation. Small but systematic differences under different experimental conditions are attributed to different times available for reaction. A method to correct for this is presented. Ne, Ar, and Kr are found to have similar energy transfer efficiencies. Complexes of cytochrome c and cytochrome b5 are detected in ESI mass spectra but with abundances less than expected from the solution equilibrium. Dissociation of the cytochrome c-cytochrome b5 complexes with charge k gives as the most abundant fragments, cytochrome b5(+3) and cytochrome c+(k-3). Adding charges to the complex destabilizes it. A series of cytochrome c variants with Lys residues thought to be involved in solution binding replaced by Ala showed no differences in the energy required to induce dissociation of the gas phase complex. The implications for the binding of the gas phase ions are inconclusive.
Mauk
M R
MR
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Mauk
A G
AG
Chen
Yu-Luan
YL
Douglas
D J
DJ
D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry; D004795:Enzyme Stability / Q000235:genetics; D008956:Models, Chemical; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D003574:Cytochrome c Group; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D015786:Cytochromes b5