# Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry
## 1 Introduction  
Electrospray ionisation (ESI) is well known for its ability to form intact protein ions for sensitive detection by mass spectrometry [1]. For large biomolecules, a key characteristic of ESI is the formation of a distribution of highly charged ions [2; 3]. This multiple charging effect has many advantages. High charging extends the effective mass range of instruments with upper \(m/z\) limits, such that proteins can be detected on essentially any type of ESI-equipped mass spectrometer [4]. For charge-sensitive mass analysers, the instrument response increases linearly with the charge state of the ion and thus, more highly charged ions can be detected with higher sensitivity and lower detection limits [5]. Moreover, protein ions that are formed with higher ion abundances and more extensive charging typically yield richer product ion spectra, corresponding to increased information regarding the sequence of the protein and any post-translational modifications. For example, in electron capture dissociation (ECD) [6; 7; 8], electron transfer dissociation (ETD) [9; 10], and some types of ultraviolet photodissociation (UVPD) [11; 12], the extent of the ion dissociation andsequence coverage can increase significantly with both the charge state and the abundance of the precursor ion. In 'top-down' MS, intact protein ions are often formed from denaturing solutions that are acidified and contain an organic modifier. Such solutions facilitate the elongation of the protein ions' conformations, which have higher surface areas and more exposed basic sites and can thus accommodate higher charge states than protein ions formed from more 'native-like' solutions [13]. However, a challenge with top-down MS is that, in ESI, charge state distributions tend to be broad, which effectively distributes the protein signal across multiple detection channels [8]. Moreover, the use of ECD, ETD, and/or UVPD can result in the formation of hundreds of product ions, further partitioning the ion signal and reducing signal-to-noise levels [14]. Thus, methods that can be used to increase the abundances of whole proteins formed by ESI are desirable. In ESI-MS, the extent of ion charging, sensitivity, and detection limits depends on many factors including solution composition, emitter size and geometry, and instrumental factors. For example, the use of chemical additives in ESI solutions have been demonstrated to increase the charge states of proteins and peptides, which can improve the efficiency of MS-based proteomic workflows, in an approach termed'supercharging' [2]. A number of different supercharging additives have been reported, including \(m\)-nitrobenzyl alcohol [15], dimethyl sulfoxide (DMSO) [16], sulfolane [17], and cyclic alkyl carbonates [7; 9; 14; 18; 19; 20] such as 1,2-butylene carbonate (C2). Our group has demonstrated that the latter class of additives can be used to form positively charged proteins in higher charge states than by use of other additives [4; 6], and such highly charged protein ions are sufficiently reactive that they can protonate Ar (g) and N\({}_{2}\) (g) in thermal, ambient temperature ion-molecule reactions [18]. [PERSON] and co-workers reported that DMSO can be doped into the eluent in LC-MS/MS to significantly increase the number of proteins and peptides identified in whole-cell digests by 10-25%, resulting in an improvement of the signal for peptide ions in bottom-up proteomics by up to ten fold [19]. The use of ESI at low flow rates (mid-to-low nL/min) and with narrow ion emitter capillaries (i.e., nanoelectrospray ionisation, nESI) in MS can be highly beneficial in the analysis of biomolecules [20; 21; 22]. The use of lower solution flow rates and narrow emitters in the range of nL/min can be used to form initial droplets that are an order of magnitude smaller than those in more conventional ESI [23; 24].