title: Site-specific analysis of gas-phase hydrogen/deuterium exchange of peptides and proteins by electron transfer dissociation.
abstract: To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.
authors.affiliation: The Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark. kdr@farma.ku.dk
authors.forename: Kasper D
authors.initials: KD
authors.lastname: Rand
authors.forename: Steven D
authors.initials: SD
authors.lastname: Pringle
authors.forename: Michael
authors.initials: M
authors.lastname: Morris
authors.forename: Jeffery M
authors.initials: JM
authors.lastname: Brown
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D003903:Deuterium / Q000032:analysis*; D041961:Deuterium Exchange Measurement; D004579:Electron Transport; D006801:Humans; D006859:Hydrogen / Q000032:analysis*; D010446:Peptide Fragments / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D025801:Ubiquitin; D006859:Hydrogen; D045304:Cytochromes c; D003903:Deuterium