Impact of charge state on gas-phase behaviors of noncovalent protein complexes in collision induced dissociation and surface induced dissociation

Abstract

Charge states of noncovalent protein complexes in the gas phase are known to affect their propensity for unfolding and dissociation. In this work, C-reactive protein (CRP) pentamer and Concanavalin A (ConA) tetramer at different charge states were subjected to collision induced dissociation (CID) and surface induced dissociation (SID) in a modified quadrupole/ion mobility/time-of-flight mass spectrometer. Charge manipulation was achieved through solution addition of charge reducing (triethylammonium acetate) or supercharging (3-nitrobenzylalcohol) reagents. The results show that charge reduction increases the stability of the proteins to dissociation and suppresses unfolding of the precursors. While CID becomes less effective at dissociation of charge reduced CRP and ConA, SID showed better preserved subunit contacts that are useful for quaternary structure elucidation. In contrast, supercharging of CRP and ConA leads to facile dissociation into subunits even for CID. The extent of precursor unfolding also increases with greater charge. Another interesting finding is that low-charge multimer products (dimers, trimers, etc.) seem to be collapsed after being released from the complexes. Further investigation is necessary to fully understand this behavior. The data presented here suggest that charge manipulation can be used to "tune" the dissociation behavior of noncovalent protein complexes in order to obtain the most useful information desired for structural analysis.

Effects of Cations on Protein and Peptide Charging in Electrospray Ionization from Aqueous Solutions

“…Low charge state ions of proteins and protein complexes are often more compact and more stable to induced conformation changes and gas phase collisional cross sections of low charge states of proteins are often similar to cross sections calculated from crystal structures [20, 74, 75]. The charge states of proteins can be reduced by several different methods, including ion-ion [76], ion-electron [77, 78] or ion-molecule reactions [21, 44].…”

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology

“…15–20 Reduction of the charge state of the precursor was shown to decrease unfolding of the proteins and resulted exclusively in observation of symmetric charge partitioning pathways consistent with the quaternary structures. 17 Complexes in higher charge states (supercharged) were more likely to unfold and exhibited less preservation of higher order structure. This phenomenon was rationalized based on ions in higher charge states having lower stabilities and greater propensity to unravel in the gas phase compared to ions in lower charge states.…”

Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein

“…22 Subsequent CIU experiments targeted protein-protein and protein-ligand complexes, and highlighted the ability of gas-phase unfolding to detect minor differences in protein stability connected to changes in both local and global protein structure. 23–27 More recent experiments have discerned the ability of CIU to detect conformationally-selective ligand binding, 28 the cooperative stabilization upon ligand binding in multiprotein complexes, 29 the details of disulfide bond structure within intact antibodies, 30 and a domain-correlated mechanism of gas-phase unfolding overall. 31 Despite these insights, we still lack a clear, detailed picture of protein CIU.…”