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

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 ¹³C and ¹⁵N 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 (c₄ - c₇, c₂₈, c₃₁, c₃₂, and c₃₃) 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.