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

Abstract

Rationale 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. Methods 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. Results 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 SO42− additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO42−. These results could reveal that the nonspecific binding was caused by electrostatic interaction. Conclusions 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.

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“…28 Meanwhile, proteins produced by nanospray ionization often bear distinct charge state distributions and the window varies with protein structure, buffer condition, instrument settings and some other parameters. Consequently, protein ions with different charge states may represent different structural populations in terms of binding behavior, 29 unfolding pathway 30 and topologies. 31,32 IM-MS can probe protein structures and conformational changes as a function of charge state, e.g.…”

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“…Meanwhile, based on the fitting curve, the specific or non-specific binding of the ligand to the protein can be also differentiated. 15 Unfortunately, there was no comparison between the direct calculation method and the curve fitting method for the same P-L binding system.…”

Comparison of the pH‐dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility‐mass spectrometry

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“…Previous ESI‐MS studies of transition metal complexes include the formation and collisional activation of His containing peptides, polyalanines, angiotensin I, oxytocin, Phe containing peptides, metalloproteins, Escherichia coli transcriptional repressor RcnR, and ubiquitin . The observation and control of nonspecific metal adduction versus specific binding has also been the attention of ESI‐MS studies …”