title: Enhancing the quality of H/D exchange measurements with mass spectrometry detection in disulfide-rich proteins using electron capture dissociation.
abstract: Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a potent technique to probe higher-order structures, dynamics, and interactions of proteins. While the range of proteins amenable to interrogation by HDX MS continues to expand at an accelerating pace, there are still a few classes of proteins whose analysis with this technique remains challenging. Disulfide-rich proteins constitute one of such groups: since the reduction of thiol-thiol bonds must be carried out under suboptimal conditions (to minimize the back-exchange), it frequently results in incomplete dissociation of disulfide bridges prior to MS analysis, leading to a loss of signal, inadequate sequence coverage, and a dramatic increase in the difficulty of data analysis. In this work, the dissociation of disulfide-linked peptide dimers produced by peptic digestion of the 80 kDa glycoprotein transferrin in the course of HDX MS experiments is carried out using electron capture dissociation (ECD). ECD results in efficient cleavage of the thiol-thiol bonds in the gas phase on the fast LC time scale and allows the deuterium content of the monomeric constituents of the peptide dimers to be measured individually. The measurements appear to be unaffected by hydrogen scrambling, even when high collisional energies are utilized. This technique will benefit HDX MS measurements for any protein that contains one or more disulfides and the potential gain in sequence coverage and spatial resolution would increase with disulfide bond number.
authors.affiliation: Department of Chemistry, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Cedric E
authors.initials: CE
authors.lastname: Bobst
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000818:Animals; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D004220:Disulfides; D011506:Proteins