Gas phase hydrogen/deuterium exchange of proteins in an ion trap mass spectrometer

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled with gas phase hydrogen/deuterium (H/D) exchange is demonstrated to be a useful tool to investigate the gas phase conformations of proteins when coupled with a mechanistic understanding of exchange. We have investigated the H/D exchange of multiple charge states of lysozyme, cytochrome c, ubiquitin, insulin, thioredoxin and melittin with deuterated methanol in the ion trap. This allows a direct comparison of exchange of these well studied proteins under identical conditions. For all proteins except lysozyme, exchange results in some peak broadening but no evidence of distinct conformers is observed. Qualitatively, trends in exchange levels are consistent with prior Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) experiments. Consistent with mechanistic studies which have shown that amine hydrogens in peptides exchange rapidly, a correlation between the number of amine hydrogens and exchange level is observed. Highest levels of exchange are observed for proteins without disulfide bonds, and for proteins which are protonated on sites other than arginine. Both of these observations are explained by the "relay" mechanism of exchange. These results indicate that a further understanding of both the dynamics of gas phase molecules and mechanisms of exchange are necessary to relate gas phase H/D exchange data more directly to protein conformation.