Journal
ANALYTICAL CHEMISTRY
Volume 85, Issue 10, Pages 5161-5168Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac4004979
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Funding
- National Science Foundation CAREER award [MCB-1149538]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1149538] Funding Source: National Science Foundation
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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 HID 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 HID exchange kinetics by an intrinsically disordered protein, murine CBP2059-2117 (UniProt CBP_MOUSE). H/D exchange data from CBP, both free and in complex with human ACTR(1018-1688) (UniProt NCOA3_HUMAN), were consistent with previous biophysical studies of this protein.
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