On the mechanism of theta capillary nanoelectrospray ionization for the formation of highly charged protein ions directly from native solutions

Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to ‘supercharge’ protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS. For θ-nanoESI, dual-channelled nanoESI emitters are used to mix two solutions in low to sub-μs prior to MS. The mechanism for θ-nanoESI mixing has been reported to occur in the Taylor cone prior to ESI-droplet formation, or by the fusion of droplets formed from separate Taylor cones. Using θ-nanoESI-ion mobility-MS, native protein solutions were rapidly mixed with denaturing supercharging solutions to form protein ions in significantly higher charge states and with more elongated structures than those formed by pre-mixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone, then the extent of protein charging and unfolding should be comparable or less than that obtained by pre-mixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. The presence of supercharging additives in pre-mixed solutions can suppress volatile electrolyte evaporation, limiting the extent of protein charging compared to when the additive is delivered via one channel of a θ-nanoESI emitter. In θ-nanoESI, the formation of two Taylor cones can presumably result in substantial electrolyte evaporation from the ESI droplets containing native-like proteins prior to droplet fusion, thereby enhancing ion charging.