What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

Abstract

Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate to those observed by ESI– MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase the extent of charging. This region incorporates properties, e.g., basicities, intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging (“supercharging”) such as m–NBA, sulfolane, and 3–nitrobenzonitrile increase analyte charge from “denaturing” and “native” solvent systems. It is suggested that additives’ Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte's solution ionization, excess spray charge is bestowed on evaporating ions carryingfewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte).

Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives

“…The addition of AAs to the spraying solution influences both the CSD and thermal stability of noncovalent protein complexes in ESI-MS. The gradual shift of protein CSD toward lower charge states with the increase in proton affinity of the AA additive (Table 1) is consistent with the mechanism recently proposed by Ogorzalek Loo et al 30 According to this mechanism, solutes with high proton affinity added at a high concentration to the protein solution can carry away a significant share of protons from the ESI droplet via the ion ejection process, thus reducing the charge available to the protein and driving the shift in protein CSD. Consistent with the mechanism proposed by Ogorzalek Loo et al and our observations, Clarke and Campopiano have recently reported a noticeable shift to lower charge states in the ESI-MS of bovine serum albumin (BSA) promoted by the addition of 5 mM Lys or His into the working protein solution (200 mM aqueous NH 4 Ac).…”

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry

“…Thus, because a denatured protein will have a much larger surface area than its native (compact) form, the denatured protein will not necessarily conform to the Rayleigh charge limit theory and may be observed with charge states greater than the Rayleigh charge ( z > z R ). (For a review on protein charging and supercharging with a focus on ESI, see [79]). Based on the results presented here and elsewhere [35, 51, 63, 73, 80], we posit that the residue fragmentation propensities for intact proteins are primarily influenced by the thermalized structures, the net charge, dissociation technique, and the residue composition of precursor ions.…”

Enhancing protein disulfide bond cleavage by UV excitation and electron capture dissociation for top-down mass spectrometry

“…These include online electrolytic reduction prior to electrospray [23–26], activation/dissociation of metal cation-adducted species [27], and radical-driven approaches [28]. Our research group previously introduced supercharging reagents to enhance charging of native proteins and complexes [29–31]. We demonstrated that by activating higher charged proteins, ECD could access more sequence information, especially for proteins containing multiple disulfide bonds [32].…”