Analysis of disordered proteins using a simple apparatus for millisecond quench-flow H/D exchange

Anal Chem. 2013 May 21;85(10):5161-8. doi: 10.1021/ac4004979. Epub 2013 May 1.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CREB-Binding Protein / chemistry*
  • Deuterium Exchange Measurement / instrumentation*
  • Humans
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Time Factors

Substances

  • Peptide Fragments
  • CREB-Binding Protein