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 with 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 carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte).

Figure 1

Three–Regime View of Electrospray Ionization for a spray operating in cone–jet mode. Region A corresponds to liquid from the edge of the emitter tip through the Taylor cone up to the jet, B corresponds to the jet and C to the point where the jet disrupts to initiate an electrospray plume. An expanded view of the plume shows stages of ESI droplet evolution, regions D, E, F, and G. As droplet D evaporates, the increasing electrostatic repulsion causing it to distort (E), and ultimately eject its own secondary droplets by asymmetric fission (F). Excess charge is disbursed to primary droplets in region C, and to secondary droplets in G. Steps D–G may repeat to produce higher order droplet progeny, until the volatile droplet evaporates. Our model assumes that ions, as well as droplets are ejected from regions C and/or G to yield H, a gas phase analyte ion (likely solvated). Our model considers that a small number of opposing charges may also be present in the gas phase ion.

Figure 2

Droplets released by a vibrating orifice aerosol generator are subjected to electric fields to induce distortion and jetting, as part of field induced droplet ionization (FIDI). (Reprinted with permission from R. L. Grimm and J. L. Beauchamp, J. Phys. Chem. B. 109, 8244–8250 (2005), copyright 2005 American Chemical Society.)

Figure 3

Subunit interactions are maintained, despite the increased charge borne by a 28-mer complex. ESI-MS of Methanosarcina thermophila 20S proteasome (690-kDa) (a) without, and (b) with 0.25% m-NBA. (Reprinted Fig. S1, with permission, from Lomeli, et al [53].

Figure 4

Supercharging agents increase charge monotonically as more and more reagent is added, whereas the onset of denaturation is marked by the sharp onset of a second distribution. ESI-MS of equine holo-myoglobin with (a) 0%, (b) 0.5% (c) 2%, and (d) 5% propylene carbonate (v/v) show smoothly increasing CSDs.

Figure 5

(a) Positive ion ESI-MS with +15 V trap-cell collision energy of equine holo myoglobin in 20 mM ammonium acetate, and (b) adding 0.5% m-NBA, and (c) with 0.5% m-NBA and +40 V trap-cell. Increasing collision energy reduces m-NBA adducts (●), increases the abundance of higher charge states, and induces heme-loss to yield apo-protein (○). Preferential clustering to high charge states of holo–myoglobin by supercharging reagents, and their detachment as neutrals, reveals that additives have low gas phase basicities (as compared to analyte) and that they interact with the protein. (Reprinted Fig. S2, with permission, from Lomeli, et al [53].

Figure 6

(a) Negative ion ESI spectra of RNase A from 50% CH₃CN/H₂O and 10 mM NH₄OAc without, and (b) with 200 mM sulfolane. (c) Positive ion ESI spectra without, and (d) with 200 mM sulfolane.