Charge-dependent modulation of specific and nonspecific protein-metal ion interactions in nanoelectrospray ionization mass spectrometry.

Abstract

Previous studies found that charge state could affect both specific and nonspecific binding of protein-metal ion interactions in nanoelectrospray ionization mass spectrometry (nESI-MS). However, the two kinds of interactions have been studied individually in spite of the problem that they often coexist in the same system. Thus, it is necessary to study the effects of charge state on specific and nonspecific protein-metal ion interactions in one system to reveal more accurate binding state. The HIV-1 nucleocapsid protein (NCp7(31-55)) which can bind specifically and nonspecifically to Zn2+ served as the model to show the charge-dependent protein-metal ion interactions. Hydrogen/deuterium exchange (HDX) and photodissociation (PD) were used to demonstrate that specific binding state was correlated with protein structure. In addition to NCp7(31-55), three other model proteins were used to investigate the reason for the charge-dependent nonspecific binding. For specific binding, we proposed that protein ions with different charge states had different conformations. The HDX results showed that labile protons in the NCp7(31-55)-Zn complex were exchanged in a charge-state-dependent way. The PD experiments revealed differential fragment yields for different charge states. For nonspecific binding, higher charge states had more Zn2+ additions, but less SO4 2- additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO4 2- . These results could reveal that the nonspecific binding was caused by electrostatic interaction. For specific binding, NCp7(31-55) with lower charge states have folding and undenatured structures. The binding states of lower charge states can better reflect more native binding states. For nonspecific binding, when multiple metal ions adduct to proteins, the proteins have more net positive charges, which tend to generate higher charge ions during electrospray.