title: Analysis of disordered proteins using a simple apparatus for millisecond quench-flow H/D exchange.
abstract: Measurement of amide H/D exchange on the ms time scale can provide valuable information about the dynamic behavior of the most flexible regions of proteins. We describe here a simple mixing apparatus, assembled solely from off-the-shelf components, that can be used for H/D exchange mass spectrometry to measure exchange on the 50-5000 ms time scale. Our apparatus utilizes flow-injection to minimize sample consumption. Although the mixer operates at low Reynolds numbers (less than 10(2)) where laminar flow is expected, H/D exchange kinetics were well-approximated using the assumption of plug-flow. We validated this approximation using fluorescence imaging of fluorescein-conjugated bovine serum albumin in the delay line and by demonstrating agreement between measured and calculated H/D exchange kinetics for a mixture of peptides. The performance of the apparatus was further validated by measuring rapid H/D exchange kinetics by an intrinsically disordered protein, murine CBP(2059-2117) (UniProt CBP_MOUSE). H/D exchange data from CBP, both free and in complex with human ACTR(1018-1088) (UniProt NCOA3_HUMAN), were consistent with previous biophysical studies of this protein.
authors.affiliation: Department of Chemistry and the Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas 66045, United States.
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Keppel
authors.forename: David D
authors.initials: DD
authors.lastname: Weis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000295:instrumentation*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D013997:Time Factors
chemical_list: D010446:Peptide Fragments; D050882:CREB-Binding Protein