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.
Charlebois
Jay P
JP
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Patrie
Steven M
SM
Kelleher
Neil L
NL
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003903:Deuterium; D004583:Electrons; D005583:Fourier Analysis; D005656:Fungal Proteins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics
D002247:Carbon Isotopes; D005656:Fungal Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D025801:Ubiquitin; D003903:Deuterium