title: Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry.
abstract: Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the  back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Sabine
authors.initials: S
authors.lastname: Amon
authors.forename: Morten B
authors.initials: MB
authors.lastname: Trelle
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
mesh_terms: D002623:Chemistry Techniques, Analytical; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D036103:Nanotechnology; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D013696:Temperature; D025801:Ubiquitin / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry
chemical_list: D011506:Proteins; D025801:Ubiquitin; D001613:beta 2-Microglobulin; D006859:Hydrogen; D003903:Deuterium