title: Strong anion exchange for studying protein-DNA interactions by H/D exchange mass spectrometry.
abstract: The use of mass spectrometry to study protein-ligand interactions is expanding into more complex systems including protein-DNA interactions. The excess amount of a model DNA or, more typically, an oligodeoxynucleotide (ODN), needed to study such interactions in an amide hydrogen-deuterium (H/D) exchange experiment, for example, causes serious signal suppression in the protein analysis. We describe here a modification of the traditional H/D exchange protocol whereby we utilize a strong anion exchange column to rapidly remove the ODN from solution before MS analysis. We showed the successful incorporation of such a column in a study of two protein-ODN systems: (1) the DNA-binding domain of human telomeric repeat binding factor 2 with a telomeric oligodeoxynucleotide and (2) thrombin with the thrombin-binding aptamer. The approach gave no appreciable difference in back-exchange compared to a method in which no strong anion exchange (SAX) is used.
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri, USA.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Sperry
authors.forename: J Micah
authors.initials: JM
authors.lastname: Wilcox
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
mesh_terms: D000838:Anions; D001665:Binding Sites; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000838:Anions; D004268:DNA-Binding Proteins; D004247:DNA