Electron capture dissociation and C-13, N-15 depletion for deuterium localization in intact proteins after solution-phase exchange

摘要

For localization of deuterium atoms after solution-phase exchange with D2O, intact proteins are often digested prior to analysis by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations associated with this approach (e.g., <70% sequence coverage and some D atom scrambling during MS/MS) were sought using intact proteins and two newer methods applied to tracking H/D exchange dynamics for the first time. Using 2-4-fold signal enhancements through depletion of C-13 and N-15 isotopes and implementing the new MS/MS technique of electron capture dissociation (ECD) yielded an increased number of c and z(.) ions observed (43 vs 25) for recombinant yeast ubiquitin (9.3 kDa). Initial determination of D atom content in consecutive c ion series (c(4) - c(7), c(28), c(31), c(32), and c(33)) was demonstrated. The improved ion signal and experiment speed combined with narrower isotopic distributions markedly increases the degree of localization and feasibility of ECD-based MS/ MS after solution-phase H/D exchange. [References: 31]