Hydrogen-Deuterium Exchange Mass Spectrometry

Native His₆-MDH (2 µM and 400 µl), thermally aggregated His₆-MDH, or sHsp/His₆–MDH complexes (both formed for 30 min at 47°C) in buffer A (50 mM Hepes, pH 7.6, 50 mM KCl, 5 mM MgCl₂, and 2 mM DTT) were incubated with 50 µl MagneHis Ni particles (Promega) for 15 min at RT. His₆-MDH (aggregated or sHsp-bound) was isolated by placing the reaction in a magnetic rack. The supernatant was subsequently removed, and the beads were washed once with buffer A. D₂O-based buffer A was added to initiate amide proton–deuteron exchange. After 30 s, the exchange reaction was quenched by adding ice-cold low-pH quench buffer (500 mM K-phosphate buffer, pH 2.2) containing pepsin (25 µg/ml; Roche). Protein was digested from the Ni particles for 1 min on ice. Then, quenched, digested samples were injected into the HPLC setup with online peptic digest and analyzed on an electrospray ionization quadrupole time-of-flight mass spectrometer (QSTAR Pulsar; Applied Biosystems) as previously described (Rist et al., 2003 (link)). Analysis of deuteron incorporation into peptides was performed by using AnalystQS software (MDS SCIEX; Applied Biosystems). The assignment of the isotope peaks and the selection of the peptides presented were done manually. Analysis of bimodal isotope distributions was performed as described previously (Ungelenk et al., 2016 (link)).