Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra.
Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.
Watt
Stephen J
SJ
Chemistry Department, University of Wollongong, New South Wales, Australia.
Sheil
Margaret M
MM
Beck
Jennifer L
JL
Prosselkov
Pavel
P
Otting
Gottfried
G
Dixon
Nicholas E
NE
D000838:Anions; D002412:Cations; D003198:Computer Simulation; D053488:DnaB Helicases / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D055672:Static Electricity
D000838:Anions; D002412:Cations; C506730:dnaB protein, E coli; D053488:DnaB Helicases