# Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry
## 1 Introduction
The use of narrower tips lowers the voltage required to initiate ESI by more effectively concentrating the electric field at the emitter tip, and it reduces sample consumption resulting in initial ESI droplets with very high surface-to-volume ratios [25; 26]. Such droplets can more readily desolvate and be transferred through narrow conductance apertures to under the vacuum of an atmospheric pressure interface to a mass spectrometer, thereby improving its sensitivity [27; 28]. Moreover, the use of nanoscale ion emitters with inner diameters of less than 1 \(\upmu\)m can significantly reduce the extent of the adduction of non-volatile salts and non-volatile molecules to protein ions [29; 30], including those of protein-ligand complexes formed from native-like solutions, which can facilitate the accurate measurement of ligand-protein binding constants [31; 32; 33]. In addition, a number of instrument modifications have been developed to increase the efficiency of the transfer of ions from atmospheric pressure to the low vacuum required for MS detection, which include different types of capillaries [34; 35; 36], skimmers [37; 38], electrodynamic ion guides [39; 40], and ion funnels [41; 42; 43; 44]. Although these strategies can be highly effective, new approaches that can be used to enhance ion signal further are desired. In ESI, typically a direct current (DC) high voltage potential is applied to the ESI solution relative to a capillary entrance to the mass spectrometer to initiate and maintain the stable formation of a plume of highly charged droplets. ESI droplets formed from DC ESI can have a natural pulse frequency (generally around 1-2 kHz) owing to the physics of the droplet formation process, which depends on the sample flow rate, applied DC voltage, and properties of the solution [45]. However, externally pulsed ESI-methods have the advantage that extremely small droplets (\(\sim\)30 fL) can be formed from relatively large capillaries (e.g., \(\sim\)11 \(\upmu\)m) compared to those formed using DC ESI [46], which can potentially enhance the sensitivity via a more efficient ion desolvation. Externally pulsed ESI can result from either pulsing a high voltage continuously through a sample solution that is flowing through a capillary emitter [47], or by applying a constant DC voltage to sequentially dispense discrete droplets of a sample solution [48; 49]. Traditionally, high frequency pulsed ESI refers to frequencies of ca. 1 kHz, whereas low frequency pulsed ESI corresponds to frequencies of 1-100 Hz [45]. In addition to externally pulsed ESI, the use of very high frequency alternating current (AC) ESI (up to 400 kHz) has been reported [50; 51; 52]. In AC ESI [50; 51; 52], and likely externally pulsed ESI, the Taylor cone can be significantly'sharper' and longer than in DC ESI, which is an effect that has been attributed to the entrainment of low mobility species in the capillary meniscus that are rapidly charged and discharged owing to the high electrophoretic mobility of protons. After multiple cycles, the low mobility species (e.g., protein ions) are enriched within the Taylor cone, which substantially elongates at a half angle (\(\sim\)12\({}^{\circ}\)) that is significantly lower than that formed by DC ESI (\(\sim\)47\({}^{\circ}\)) [53; 54]. Although the optimal conditions for DC ESI generally resulted in higher ion abundances than those of AC ESI--owing primarily to the far lower electrical breakdown limit of AC vs. DC for the same maximum applied voltage [50]--the formation of a sharper Taylor cone should result in the production of smaller initial ESI droplets than those formed by DC ESI and less radial dispersion of the resulting aerosol plume. These two effects should in principle improve the efficiency of ESI-MS particularly for narrower bore nESI emitters in which the electrical breakdown limit (<1 kV) is substantially lower than that of larger bore ion emitters.