rFVIIIFc, rFVIII, and rFc were dialyzed against 10 mM histidine, pH 7.0, 5 mM CaCl2, 200 mM NaCl and 13.3 g/L sucrose. Deuterium exchange was initiated by diluting each sample ten-fold with deuterated buffer (99.99% D2O; Cambridge Isotope Laboratories, Andover, MA) to a final volume of 25 μL. After 10 s, 1 min, 10 min, 1 h and 4 h of incubation, the reaction was quenched, and the protein was denatured and reduced by addition of ice-cold quench solution (1:1, v:v) containing 7.5 M guanidinium hydrochloride, 0.2 M Tris (2-carboxyethyl) phosphine (TCEP) and 0.5 M citric acid, resulting in a pH of 2.3. This preparation was digested on an immobilized pepsin column (Life Technologies, Carlsbad, CA) inside a Waters HDX manager with the temperature maintained at 0°C. Eluted peptides were desalted, separated on an HSS T3 C18 HPLC column and introduced into a Synapt G2S mass spectrometer by electrospray ionization. Mass spectra were collected in triplicate for each exchange period. Peptides were identified by ProteinLynx Global Server (Waters, Milford, MA).
HDX data analysis was performed with the DynamX software package (Waters, Milford, MA) and statistical relevance was based on published criteria [25 (link)].
HDX data analysis was performed with the DynamX software package (Waters, Milford, MA) and statistical relevance was based on published criteria [25 (link)].
