Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor
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Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor. / Comamala, Gerard; Wagner, Cornelia; de la Torre, Pablo Sanz; Jakobsen, Rasmus U; Hilger, Maximiliane; Brouwer, Hendrik-Jan; Rand, Kasper D.
In: Analytica Chimica Acta, Vol. 1115, 08.06.2020, p. 41-51.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor
AU - Comamala, Gerard
AU - Wagner, Cornelia
AU - de la Torre, Pablo Sanz
AU - Jakobsen, Rasmus U
AU - Hilger, Maximiliane
AU - Brouwer, Hendrik-Jan
AU - Rand, Kasper D
N1 - Copyright © 2020 Elsevier B.V. All rights reserved.
PY - 2020/6/8
Y1 - 2020/6/8
N2 - Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.
AB - Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.
U2 - 10.1016/j.aca.2020.04.014
DO - 10.1016/j.aca.2020.04.014
M3 - Journal article
C2 - 32370868
VL - 1115
SP - 41
EP - 51
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
SN - 0003-2670
ER -
ID: 240836808