On the Mechanism of Theta Capillary Nanoelectrospray Ionization for the Formation of Highly Charged Protein Ions Directly from Native Solutions

Abstract

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-channeled 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 primarily occur: (i) in a single shared Taylor cone and in the droplets formed from the Taylor cone or (ii) by the fusion of droplets formed from two 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 premixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone and in the droplets resulting from the single Taylor cone, then the extent of protein charging and unfolding should be comparable to or less than that obtained by premixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. These data also suggest that highly charged protein ions can be formed by the near-complete mixing of each solution. The presence of supercharging additives in premixed 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.

Variable Mixing with Theta Emitter Mass Spectrometry: Changing Solution Flow Rates with Emitter Position

“…Prior differences in theta mixing vs premixing solutions when one solution contains a protein in a native-like conformation and the other solution contains a supercharging reagent have been attributed to the formation of two different Taylor cones and independent electrospray plumes of the two different solutions with droplets merging to initiate reaction . Our results, along with prior results on supercharging with theta glass emitters, show that if droplets combine to form mixed solutions, the efficiency of the droplet recombination would have to be 99+% in order to not observe low charge states of proteins formed from aqueous droplets containing protein that do not recombine.…”

“…While it is possible for some droplets to fuse after their initial production, there is an alternative explanation that more likely accounts for the prior results on supercharging with theta glass emitters. In the prior experiments, the relative flow rates of the two channels were not apparently monitored . As our results demonstrate, the relative flow rates of the two channels are a strong function of the emitter tip position.…”

“…44,45 A recent report by Brown et al of protein supercharging with water/methanol/1,2-butylene carbonate/acetic acid using theta glass emitters indicated that the charge-state distributions from theta glass mixing were different than those formed with premixed solutions. 28 From the subtle differences in charge state, the authors concluded that the two barrels formed independent Taylor cones and that mixing occurs by merging collisions of the progeny droplets that are both highly charged and have the same polarity. Multiple Taylor cones can occur at high electrospray potentials and with very large diameter theta glass emitters.…”

“…Mass spectrometry is widely used to study chemical reactions owing to its high sensitivity, specificity, and analysis speed. Reactions can be initiated by mixing two solutions containing reagents prior to the electrospray ionization (ESI) process, including with online mixing devices. , Reactions can also be initiated after droplet formation occurs, as is the case with droplet fusion, reactive desorption ESI, , or through introduction of gaseous reactants. , Mixing has also been done immediately prior to where droplet formation occurs using microfabricated devices , or with theta glass emitters that have two channels formed by a center septum, the end of which looks like the Greek letter θ when viewed head on. − Theta glass emitters have been used in conjunction with mass spectrometry to measure reaction intermediates, , noncovalent complexation formation, , reduction–oxidation reactions, hydrogen–deuterium exchange, , and initiate protein folding ,, or unfolding. ,, Supercharging reagents have been used with theta glass emitters to increase protein charge states. , Theta glass emitters have also been used to extract material from cells into one channel and react the cellular extract with acid or derivatizing reagents contained in the other channel prior to ESI …”

“…17,21,22 Supercharging reagents have been used with theta glass emitters to increase protein charge states. 23,28 Theta glass emitters have also been used to extract material from cells into one channel and react the cellular extract with acid or derivatizing reagents contained in the other channel prior to ESI. 24 One advantage of using theta glass emitters for mixing experiments is that mixing times are fast and the time for a solution reaction to occur can be varied by changing the initial droplet size 16,25 or by mixing solutions at the end of the capillary prior to initiation of ESI.…”