# Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry
## 3 Results
### Effects of Frequency and Pulse  
In Figure 3, mass spectra of Myo that were obtained by conventional direct current nESI and pulsed nESI at a frequency of 50, 100, 200, and 300 kHz are shown. To investigate the effects of the waveforms used in pulsed nESI, mass spectra were first obtained at a frequency ranging from 10 to 350 kHz using a fixed duty cycle of 50% to determine the 'optimal' frequency (Figure 4a). With increasing frequency from 10 kHz, the absolute ion abundance of Myo generally and steadily increased to a maximum at 200 kHz and then decreased as the frequency increased further to 350 kHz. Mass spectra were then obtained using the optimal frequency, but with different duty cycles. The signal intensity increased as the duty cycle increased, resulting in a peak at a duty cycle of 60% followed by a gradual decrease with higher duty cycles (i.e., \(\geq\)70% to up to 90%; Figure 4c). The optimal frequency of 200 kHz and duty cycle of 60% were used to collect all other proteins'  
Figure 1: Diagram of pulsed nanoelectrospray ionization mass spectrometry. (**a**) A borosilicate capillary emitter pulled to an inner diameter of \(\sim\)250 nm. The emitter was filled with a sample solution (blue); a thin platinum wire was inserted to apply the spray voltage into the solution. (**b**) A pulsed, positive polarity high voltage potential was applied to the emitter relative to the capillary entrance to the mass spectrometer (MS), which had sub-nanosecond rise times, pulse widths (\(T_{\text{P}}\)) from 2.85 to 100 μs, and a delay between pulses (\(T_{\text{S}}\)) of 0.25 to 90 μs. spectra and compared to those collected from the conventional direct current nESI method (Figure 2). The use of pulsed nESI resulted in an increase in the average charge state distributions of the two larger protein ions (Figures 2 and S2, Table 1). For example, the average charge state distribution of Myo increased from 19.5 \(\pm\) 0.07 to 20.6 \(\pm\) 0.09 as the frequency increased from 10 to 200 kHz and then decreased slightly as the frequency decreased further (Figure S2). Likewise, the most abundant charge state of Myo shifted from the 20+ for DC nESI to the 23+ for pulsed nESI at 200 kHz (Figure 2c,g). For CAII, the average charge state increased slightly from 36.5 \(\pm\) 0.47 for DC nESI to 37.5 \(\pm\) 0.44 for pulsed nESI at 200 kHz (Table 1). Such a shifting to a higher charge state distribution at a higher frequency is consistent with results reported previously for protein ions formed from denaturing solutions using AC and pulsed ESI [52; 55]. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Analyles**} & \multicolumn{3}{c}{\(N_{\text{C}}\) *} & \multicolumn{3}{c}{**S/N\({}_{\text{C}}\) *} & \multicolumn{3}{c}{**Average Charge State*****} \\ \cline{2-9}  & **Pulsed nESI** & **nESI** & **\% Decrease *b** & **Pulsed nESI** & **nESI** & **\% Increase *b** & **Pulsed nESI** & **nESI** \\ \hline Ubq & \(105\pm 3.99\) & \(155\pm 10.5\) & 32.5\% & \(2334\pm 71.4\) & \(1406\pm 117\) & 66.0\% & \(12.1\pm 0.54\) & \(11.9\pm 0.33\) \\ CytC & \(206\pm 52.2\) & \(273\pm 35.2\) & 24.4\% & \(1313\pm 143\) & \(615\pm 66.2\) & 114\% & \(15.6\pm 0.42\) & \(15.9\pm 0.40\) \\ Myo & \(158\pm 9.63\) & \(185\pm 11.3\) & 14.6\% & \(709\pm 23.9\) & \(461\pm 17.9\) & 53.8\% & \(22.0\pm 0.34\) & \(20.6\pm 0.36\) \\ CAII & \(272\pm 48.0\) & \(504\pm 102\) & 46.1\% & \(518\pm 128\) & \(204\pm 66.9\) & 154\% & \(37.5\pm 0.44\) & \(36.5\pm 0.47\) \\ Heme & \(148\pm 7.63\) & \(181\pm 9.31\) & 18.2\% & \(124\pm 8.32\) & \(891\pm 5.91\) & 39.2\% & N/A * & N/A * \\ Ang & \(92.1\pm 7.31\) & \(165\pm 8.37\) & 44.2\% & \(3943\pm 103\) & \(2082\pm 24.7\) & 89.3\% & \(2.08\pm 0.09\) & \(1.97\pm 0.10\) \\ \hline \hline \end{tabular}  
* Values correspond to the mean \(\pm\) standard deviation of at least three replicate measurements; \({}^{b}\)_p_-value \(<\) 0.05; \({}^{c}\) Not applicable.  
\end{table}