Abstract
A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.
Highlights
Hydrogen/deuterium exchange (HDX)1 measured by mass spectrometry (HDX-MS) is a powerful tool to probe the structure and dynamics of proteins in solution [1,2,3,4]
On-line Deuteration of a Short Peptide Using the HDX Cell—To test the feasibility of on-line deuteration for HDX-MS, we carried out on-line labeling of [Glu1]-fibrinopeptide B, a 14-residue peptide with no known secondary structure elements
To investigate the effect of D2O flow rate on HDX cell deuteration efficiency, [Glu1]-fibrinopeptide B was infused at a constant flow rate of 0.5 l/min whereas that in the channel carrying deuterium oxide was varied between 0.1 and 10 l/min
Summary
Hydrogen/deuterium exchange (HDX)1 measured by mass spectrometry (HDX-MS) is a powerful tool to probe the structure and dynamics of proteins in solution [1,2,3,4]. We describe a method for dilution-free on-line deuterium labeling of proteins using an HDX cell with two flow channels separated by an ion-selective membrane [7]. To probe the effect of deuterium oxide (D2O) flow rate on the deuterium ion content in the sample channel, the setup can be operated with or without addition of quenching solution (30% acetonitrile and 0.1% formic acid, pH 2.4) (supplemental Fig. S1).
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