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Analysis of Disordered Proteins Using a Simple Apparatus for Millisecond Quench-Flow H/D Exchange
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...
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Published in: | Analytical chemistry (Washington) 2013-05, Vol.85 (10), p.5161-5168 |
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Main Authors: | , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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 102) 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 CBP2059–2117 (UniProt CBP_MOUSE). H/D exchange data from CBP, both free and in complex with human ACTR1018–1088 (UniProt NCOA3_HUMAN), were consistent with previous biophysical studies of this protein. |
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ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/ac4004979 |