Effects of electrospray capillary temperature on amide hydrogen exchange

Amide hydrogen/deuterium (H/D) exchange coupled with proteolysis, high-perfeomance liquid chromatographic (HPLC) separation and mass spectrometry (MS) has become a powerful tool to study protein dynamics in solution. Prior to the execution of H/D exchange experiments, various experimental parameters have to be set, including proteolysis, HPLC, and MS conditions. Here we investigate the effects of electrospray capillary temperature on deuterium retention in backbone amides of various pepsin-generated cytochrome c peptides. Lower capillary temperature generally helps retain more deuterium than higher capillary temperature. When the capillary temperature was 150 degrees C, on average 26% more deuterium was retained than when the capillary temperature was set at 250 degrees C. The effects of capillary temperature varied depending on the ions monitored. There was little difference in deuterium retention among different charge state species of the same peptide at 150 degrees C. However, a lower charge state ion loses more deuterium atoms going from 150 degrees C to 250 degrees C than the corresponding higher charge state species. These results indicate that the capillary temperature should be optimized not only to maximize the signal-to-noise of each ion followed in H/D exchange experiments, but also to minimize the deuterium loss of the ions. Also the loss of deuterium in several ions, especially lower charge state ones, should be monitored in the optimization, as the temperature effects vary among ions and are more significant for lower charge state ions.