Highly Charged Protein Ions: The Strongest Organic Acids to Date

Abstract

The basicity of highly protonated cytochrome c (cyt c) and myoglobin (myo) ions were investigated using tandem mass spectrometry,i on-molecule reactions (IMRs), and theoretical calculations as af unction of charge state. Surprisingly,h ighly charged protein ions (HCPI) can readily protonate non-polar molecules and inert gases,i ncluding Ar, O 2 ,a nd N 2 in thermal IMRs.T he most HCPIs that can be observed are over 130 kJ mol À1 less basic than the least basic neutral organic molecules known( tetrafluoromethane and methane). Based on theoretical calculations,itispredicted that protonated cyt ca nd myo ions should spontaneously lose ap roton to vacuum for charge states in which every third residue is protonated. In this study,H CPIs are formed where every fourth residue on average is protonated. These results indicate that protein ions in higher charge states can be formed using al ow-pressure ion source to reduce proton-transfer reactions between protein ions and gases from the atmosphere.Electrospray ionization (ESI) is renowned for its ability to form intact, gaseous,m ultiply charged protein ions for rapid and sensitive detection by mass spectrometry. [1] However,the mechanism by which protein ions are formed in ESI is controversial and continues to be actively debated. Thet wo primary competing models to explain ion formation are known as the charge residue model (CRM) [2] and the ion evaporation model (IEM). [3] In both models,a sn eutral molecules evaporate from ac harged droplet, the electric field at the surface of the droplet increases,w hich initiates fission. [4] Such droplet fission events result in the emission of afine stream of smaller droplets that remove less than 1% of the mass but more than 30 %o ft he charge of the precursor droplet. [4,5] In the CRM, sequential droplet evaporation and Coulombic fission events yield acharged droplet that contains asingle analyte ion, which evaporates to dryness via the loss of neutral solvent molecules.Inthe IEM, the electric field on the surface of ah ighly charged droplet near the moment of ion formation is sufficient to result in the ejection of an analyte ion from the surface of the ionic droplet. Themajority of current evidence indicates that fully desolvated protein ions formed from buffered aqueous solutions are formed by the CRM. Charge carriers such as solvated hydronium ions can be lost via ion evaporation during the ESI process. [6] Recently,the chain-ejection model (CEM), [7] which is related to the IEM, was proposed to explain the formation of protein ions from denaturing solutions based on results from molecular dynamics simulations. [7] In the CEM, ad enatured, disordered protein chain is ejected from ah ighly charged, nanometer-sized ionic droplet. As the protein ion protrudes and is ejected from the droplet, proton transfer to the protein ion can occur. However,t he mechanism by which highly charged protein ions (HCPIs) are formed from denaturing solutions is less well established with evidence supporting both the CRM [8] and CEM/IEM having be...

Metal Ion Binding to the Amyloid β Monomer Studied by Native Top-Down FTICR Mass Spectrometry

“…The fact that the chelating agent is lost as a protonated species under these conditions is perhaps surprising; however, loss of a 3− anion from a 7+ cation is likely to be energetically unfavorable. It has been shown previously that multiply charged protein cations can act as surprisingly strong proton donors in the gas phase due to electrostatic repulsion between protons [58]. Interestingly, further activation of this ion resulted in backbone cleavage rather than the observation of any iron-free amyloid ( vide infra ), indicating that the gas-phase interaction between Fe 3+ and the amyloid was strong, consistent with our hypothesis that solubility was the limiting factor in the initial experiments.…”

Supercharging Proteins: How Many Charges Can a Protein Carry?

“…Ther ole of supercharging reagents in destabilizing folded structures,aswell as increasing the surface tension of droplets (leading to higher-charged droplets before Coulomb fission occurs), are both compatible with increasing charge states in proteins, [2d] though direct interactions between the proteins and reagents may also play arole. [2b] In the experiments by Donald and co-workers, [3] supercharged protein charge states are trapped inside an ion trap in the presence of ac ontrolled gas environment of He,A r, O 2 and/or N 2 .I ti so bserved that the rate of deprotonation is seemingly related to the gas-phase basicity of the background gas.While deprotonation in He is slow,itismuch faster in the atmospheric gases O 2 and N 2 .T his strongly suggests that ap roton transfer takes place from the protein to the background gas.Amore definite proof would be the appearance of protonated N 2 ,O 2 ,e tc. ;h owever,s uch low masses cannot be trapped in their ion trap.…”

“…Given these advantages,e fforts in the past decade and ahalf have aimed at increasing the charge states of proteins,in so-called "supercharging" experiments. [2] This raises the intriguing question what fundamental limitations there are to attaching charges,inthis case protons,toproteins.Arecent Communication by Donald and co-workers [3] presents evidence that highly-charged protein ions are so acidic that they can lose protons to N 2 ,O 2 ,a nd even He!G iven that electrospray is effected at atmospheric pressure,o ften using N 2 drying gas,i ts eems remarkable then that such highlycharged species can even be formed, and how mechanistically one could explain it. Before we consider the factors that are thought to limit charging,l et us briefly review some of the underlying assumptions and hypotheses.…”

Electrospray Ionization-Mass Spectrometry of Synthetic Polymers Functionalized with Carboxylic Acid End-Groups

“…The CEM describes the sequential release of molecular chains by continuous addition of charges that are repelled from the charged surface of the droplet. Molecular dynamics simulations and companion experimental studies support the CEM as a viable ionization route for denatured proteins. , Currently, however, it remains unclear how these models can be applied to understanding and predicting the ionization of low-charging, nonpolar polymer chains.…”

“…Molecular dynamics simulations and companion experimental studies support the CEM as a viable ionization route for denatured proteins. 54,55 Currently, however, it remains unclear how these models can be applied to understanding and predicting the ionization of low-charging, nonpolar polymer chains.…”