Highly deuterated peptides (Waters MassPREP Peptide Standard containing RASG-1, bradykinin, and angiotensin I and II) were prepared by dissolving the lyophilized peptides into D2O that was adjusted to pD 2.5 with DCl. Peptides were allowed to deuterate at 20 °C for two hours before infusion directly into the instrument in 50:50 D2O:acetonitrile using a syringe pump.
Labeled cytochrome c (462 µM stock solution in 20 mM Tris, 100 mM NaCl and 3 mM DTT) was diluted to usable concentrations of 64 and 12.8 µM for HPLC and UPLC, respectively. Deuterium exchange was initiated by adding a 15-fold excess of 99% deuterium oxide buffer (20 mM Tris, 100 mM NaCl and 3 mM DTT) at 21 °C. At each exchange-in time point an aliquot (100 picomoles for HPLC, 20 picomoles for UPLC) from the exchange reaction was transferred to a separate tube containing an equal volume of quench buffer (300 mM potassium phosphate, pH 2.6, H2O). Quenched samples were immediately analyzed. Highly deuterated cytochrome c was prepared by diluting the stock solution 15-fold into D2O pD 2.5, incubating at 37 °C for 6 hours and quenching as described above.
Labeled cytochrome c (462 µM stock solution in 20 mM Tris, 100 mM NaCl and 3 mM DTT) was diluted to usable concentrations of 64 and 12.8 µM for HPLC and UPLC, respectively. Deuterium exchange was initiated by adding a 15-fold excess of 99% deuterium oxide buffer (20 mM Tris, 100 mM NaCl and 3 mM DTT) at 21 °C. At each exchange-in time point an aliquot (100 picomoles for HPLC, 20 picomoles for UPLC) from the exchange reaction was transferred to a separate tube containing an equal volume of quench buffer (300 mM potassium phosphate, pH 2.6, H2O). Quenched samples were immediately analyzed. Highly deuterated cytochrome c was prepared by diluting the stock solution 15-fold into D2O pD 2.5, incubating at 37 °C for 6 hours and quenching as described above.
