Investigation of amide hydrogen back-exchange in Asp and His repeats measured by hydrogen (~1H/~2H) exchange mass spectrometry

摘要

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His_6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp _6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K_2D_6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K_2D_6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling.