Supercharging of Proteins by Salts during Polarity Reversed Nano

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Supercharging of Proteins by Salts during Polarity Reversed Nano-Electrospray Ionization Xiaoyun Gong, Chang Li, Rui Zhai, Jie Xie, You Jiang, and Xiang Fang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02759 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Analytical Chemistry

Supercharging of Proteins by Salts during Polarity Reversed Nano-Electrospray Ionization Xiaoyun Gong*, Chang Li, Rui Zhai, Jie Xie, You Jiang, Xiang Fang* Division of Chemical Metrology and Analytical Science, National Institute of Metrology, Beijing, 100029, China ABSTRACT: Supercharging is beneficial in many ways to the analysis of proteins by mass spectrometry (MS). In this work, a novel supercharging method was developed. It made use of our previously developed ionization technique, namely polarity reversed nanoelectrospray ionization (PR-nESI) for the ionization of proteins. Supercharging of proteins was achieved by just adding 1−10 mM salt in sample, such as sodium chloride (NaCl). The charge state of proteins obtained by our method was significantly higher than that by nanoESI with 1% (v/v) acetic acid (HAc). Different kinds of salts were investigated. Salts with strong acid anions were capable of supercharging proteins, including chlorides, bromides, iodides and nitrates. The signal intensity and signal-to-noise ratio (S/N) of proteins were increased at the same time. Phosphates were also found to have supercharging effect, due to the fact that phosphoric acid was a mediumstrong acid. In comparison, salts with weak acid anions had no supercharging effect, such as carbonates, sulfides, acetates and formates. The species of the salt anion was critical to the supercharging effect, while the species of the salt cation showed little influence on the supercharging effect. Investigations were made into the mechanism of our method. The supercharging effect was caused by the interactions between protein molecules and salt anions, as well as the influence of protons. The present work offered us an alternative way for the supercharging of proteins. The use of common salts for supercharging made it more convenient. The concentration of salts needed for supercharging was much lower than those conventionally used for supercharging reagents. Taking into consideration of the fact that many biological samples were buffered with phosphates and chlorides, these samples could directly be supercharged by our method without any additional additives. Furthermore, many salts were non-toxic and could easily be found in a chemical lab, the use of salts for supercharging would be a much more practical and economic choice. In addition, the present work also furthered our understandings about the mechanism of supercharging, as well as electrospray.

INTRODUCTION The formation of multiply charged protein ions by electrospray ionization (ESI) is considered to be advantageous to mass spectrometry (MS) based analysis.1-3 It extends the effective mass range of many types of mass analyzers due to the reduced mass-to-charge ratios (m/z) of proteins.4 More proteins can readily be detected by mass spectrometers with a limited m/z range.5-7 Furthermore, the sensitivity and resolution of most mass spectrometers improve at lower m/z. Higher charged protein ions are more efficiently detected, especially by charge sensitive detectors, such as those in orbitrap and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers.8 Besides, protein ions with higher charge states are typically less likely to form adducts with metal ions or phosphates.9-11 This avoids the broadening of spectral peaks caused by unresolved adducts, ensuring an improved sensitivity and mass accuracy. 12,13 In addition, tandem mass spectrometry (MS/MS) is generally more efficient and structurally informative on higher charged precursor ions.14-17 This phenomenon is especially remarkable when electron capture dissociation (ECD) 18,19 and electron transfer dissociation (ETD) 20-22 are carried out. Several methods have been developed to increase the charge state of protein ions generated from ESI. Commonly, increasing protein charge is achieved by using denaturing

solutions. These solutions typically contain high concentrations of acids or bases to regulate the pH. 23 Protein molecules are unfolded under such pH, resulting in an increased charge state. Organic solvents might also be added to solutions to assist the unfolding and increasing charge of proteins.5,24 High charge-state ions can also be generated from unbuffered electrospray droplets by introducing acid or base vapors into the drying gas of the ESI interface. 25,26 The pH of the droplet is changed by the acid or base vapors during desolvation, resulting in the unfolding and supercharging of protein molecules. The addition of supercharging reagents in solutions offers an alternative way to increase the charge state of proteins. These reagents work well in both native and denatured solutions. The most commonly used supercharging reagents are m-nitro benzyl alcohol (m-NBA)6,7,15,27-31 and sulfolane28,32-34 and dimethyl sulfoxide (DMSO)35,36. Supercharging reagents have a relatively high boiling point, thus they get enriched during the desolvation process of ESI. 36 High concentrations of supercharging reagents have considerable impact on the surface tension of the droplet, as well as the conformation of proteins. The mechanism of the supercharging process has been debated and discussed thoroughly in literature. 37 An intermediate regime hypothesis was proposed, which fully explained the results obtained in those supercharging events.

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The supercharging reagents have already been applied to liquid chromatography tandem mass spectrometry (LCMS/MS).35,38-41 The addition of certain amount of supercharging reagents directly into the mobile phase, or postcolumn into the ESI source, is shown to improve peptide and protein identification in large-scale applications. More recently, electrothermal supercharging was developed for the alteration of protein charge states. 42-45 In this method, solutions are buffered with 100 mM ammonium bicarbonate (NH4HCO3) at a neutral pH of 7.0. Low charge states of proteins and intact complexes are obtained at low ESI spray voltages, while high charge states are obtained at high spray voltages. Low and high charge states can be switched reversibly by changing the spray voltage. The mass spectra obtained by electrothermal supercharge are similar to those obtained under native and denatured conditions. Recently, polarity reversed nano-electrospray ionization (PR-nESI) was developed by us for the improvement of nano-ESI performance.46 It remarkably increased the signal to noise ratio of proteins and effectively reduced metal ion adducts. In this work, we demonstrate that with the presence of certain salts in solution, such as sodium chloride (NaCl), supercharging of proteins could easily be achieved during PR-nESI. The obtained charge state of proteins was even higher than that obtained by using conventional denaturing additives, such as acids. The concentration of salts needed for supercharging was within the range of 1−10 mM, which was much lower than those additives used in other supercharging methods. Taking into consideration of the fact that many biological samples were buffered with phosphates and chlorides, these samples could directly be supercharged by our method without any additional additives. Furthermore, many salts were non-toxic and could easily be found in a chemical lab, the use of salts for supercharging would be a much more practical and economic choice. Although the presence of salts would cause severe signal suppression in conventional nano-ESI, the signal obtained by PR-nESI was not affected at all. Few metal ion adducts were observed. Further investigations were made into the mechanism of the supercharging effect of salts during PR-nESI. The anions of the salts were critical to the supercharging effect, while the species of the cations had little impact on the supercharging effect. The present work offered us an alternative way for the supercharging of proteins. It also furthered our understandings about the mechanism of supercharging, as well as electrospray.

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tive polarity was used for the detection, a positive high voltage of +3.5 kV was first applied to the electrode for about 5−10 seconds. Afterwards, the polarity of the high voltage was reversed, and a negative high voltage of −1.35 kV was applied to the electrode to generate electrospray. To achieve the reversal of the high voltage, the entire MS was subject to polarity reversal. Except for the high voltage strategy, the other settings of PR-nESI were the same as nano-ESI. All of the proteins were detected in positive polarity mode in this work. When carrying out conventional nano-ESI, a spray voltage of +1.75 kV was used for positive polarity and a spray voltage of −1.35 kV was used for negative polarity. Supercharging of Proteins. Proteins were dissolved in ultrapure water in a concentration of 5−15 μM. Salts were added to the solutions in a concentration of 1−10 mM. The obtained solutions were detected by PR-nESI. Peaks representing supercharged protein molecules were observed in the obtained mass spectra. The other experimental details were described in the Supporting Information.

RESULTS AND DISCUSSION Detection of Different Proteins by NaCl. Different proteins were investigated to confirm the supercharging capability of NaCl during PR-nESI, including cytochrome c (Cyt c), holomyoglobin (holo-Mb), calmodulin (CaM) and lysozyme (Lys) (Figure 1). A representative nano-ESI mass spectrum of Cyt c was shown in Figure 1a. Cyt c was dissolved in ultrapure water. The obtained solution was directly detected by nano-ESI. A unimodal CSD of Cyt c was observed. It ranged from 7+ to 11+ and centered at 8+. The intensity-weighted average charge state (qav) of Cyt c was included in the figure inset. It was calculated according to Equation 1, in which qi was the net charge of the ith charge state, Wi was the signal intensity of the corresponding ith charge state, and N was the total number of the observed protein charge states in the spectrum.29 The qav of Cyt c was 8.2+. The result obtained by PR-nESI with the presence of 5 mM NaCl in sample was shown in Figure 1b. Cyt c was dissolved in ultrapure water. 5 mM NaCl was added to the solution. The obtained solution was detected by PR-nESI. A unimodal CSD was observed. It ranged from 9+ to 22+ and centered at 18+. The qav was increased to 16.7+. 𝑞𝑎𝑣 =

∑𝑁 𝑖 𝑞𝑖 𝑊𝑖 ∑𝑁 𝑖 𝑊𝑖

(1)

The charge state of Cyt c obtained by our method was even higher than that obtained by nano-ESI with 1% (v/v) acetic acid (HAc) in solution (Figure S1a). The supercharging effect of NaCl in PR-nESI was dependent on the concentration of NaCl. 5 mM NaCl offered an optimized performance for 15 μM Cyt c sample. Further increasing or decreasing the concentration of NaCl would result in a reduced charge state of Cyt c. The combination of NaCl and PR-nESI was essential for the supercharging of Cyt c. No supercharging effect was observed under nano-ESI whether with or without 5 mM NaCl in sample (Figure S1b and Figure 1a). Na+ adducts were identified with high abundances under these two conditions. When PR-nESI was used for the detection of Cyt c

EXPERIMENTAL SECTION Generation of PR-nESI. The generation of PR-nESI was achieved according to the instructions described in our previous work.46 Briefly, a polarity-reversing high voltage strategy was used for the generation of PR-nESI. When positive polarity was used for the detection, a negative high voltage of −3.0 kV was first applied to the electrode for about 5−10 seconds. Afterwards, the polarity of the high voltage was reversed, and a positive high voltage of +1.75 kV was applied to the electrode to generate electrospray. When nega-

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with no additive in solution, no supercharging effect was observed either (Figure S1c). When 1% HAc was added to the solution, the result obtained by PR-nESI (Figure S1d) was quite similar to that obtained by nano-ESI (Figure S1a).

Figure 1. Detection of different proteins by NaCl, including Cyt c, holo-Mb, CaM and Lys. The concentrations of Cyt c, holo-Mb, CaM and Lys in samples were 15 μM, 10 μM, 5 μM and 20 μM, respectively. Representative nano-ESI mass spectra of Cyt c, holo-Mb, CaM and Lys were shown in (a), (c), (e) and (g), respectively. Proteins were dissolved in ultrapure water. The obtained solutions were directly detected by nano-ESI. The detection results of Cyt c, holo-Mb, CaM and Lys obtained by PR-nESI with the presence of 5 mM NaCl in samples were shown in (b), (d), (f) and (h), respectively. Proteins were dissolved in ultrapure water. 5 mM NaCl was added to the solutions. The obtained solutions were detected by PR-nESI.

Similar results were obtained on holo-Mb. When nano-ESI was used for the detection of holo-Mb, two CSD were observed in the spectrum (Figure 1c). The two CSD represented holo-Mb and apo-Mb, respectively. Holo-Mb had a higher abundance. The CSD of holo-Mb ranged from 9+ to 17+ and centered at 13+. The qav of holo-Mb was 13.0+. Apo-Mb had a lower abundance. The CSD of apo-Mb also ranged from 9+ to 17+ and centered at 13+. The qav of apo-Mb was 13.1+. When PR-nESI was used for the detection of holo-Mb with the presence of 5 mM NaCl in sample, the CSD of apo-Mb shifted to a much higher charge state (Figure 1d). It ranged from 12+ to 27+ and centered at 22+. The qav of apo-Mb was increased to 21.2+. No signal of holo-Mb was observed.

When nano-ESI was used for the detection of CaM, a unimodal CSD of apo-CaM was observed (Figure 1e). It ranged from 13+ to 24+ and centered at 19+. The qav of apo-CaM was 20.4+. No holo-CaM was observed. When PR-nESI was used for the detection of CaM with the presence of 5 mM NaCl in sample, a unimodal CSD of apo-CaM was also observed (Figure 1f). The CSD ranged from 12+ to 29+ and centered at 20+. The qav of apo-CaM was 20.2+. No holo-CaM was observed. The qav of apo-CaM obtained by nano-ESI was slightly higher than that obtained by PR-nESI, which implied that no supercharging of CaM was achieved by PR-nESI. This was due to the abnormal property of CaM during electrospray. It got highly charged even if the solution was buffered with 5 mM ammonium acetate (NH4Ac) (Figure S2a). The CSD of apo-CaM ranged from 10+ to 28+ and centered at 20+. The qav of apo-CaM was 20.0+. Although a high charge state was obtained under this condition, severe Na + adducts were observed. This was not favourable to the identification of the peaks. When 56.8 mM HAc was added to the solution, the CSD of apo-CaM shifted to a lower charge state (Figure S2b). The addition of 56.8 mM HAc to the solution offered a pH of 3.0, which was measured by a pH meter. The pH was lower than that offered by the commonly used 0.1% HAc (pH = 3.2). The CSD of apo-CaM ranged from 10+ to 24+ and centered at 18+. The qav of apo-CaM was decreased to 17.8+. Although the charge state was reduced, a very clean spectrum of protonated apo-CaM ions was obtained. This was advantageous to the identification of the peaks. Compared with the results shown in Figure S2, our method offered an optimized result (Figure 1f). A high charge state was obtained and no metal ion adduct was observed. The results obtained on Lys also confirmed the supercharging effect of NaCl (Figure 1g and h). When using nanoESI for the detection of Lys, the unimodal CSD ranged from 8+ to 11+ and centered at 9+. The qav of Lys was 9.0+. When PR-nESI was used for the detection of Lys with the presence of 5 mM NaCl in sample, the CSD shifted to a higher charge state. It ranged from 8+ to 12+ and centered at 10+. The qav was increased to 10.0+. The Concentration of NaCl. The concentration of the salt used in sample was crucial to the supercharging effect. To further look into this issue, different concentrations of NaCl were tried for the supercharging of Cyt c (Figure S3). A bimodal CSD was observed when the concentration of NaCl was 1 mM (Figure S3a). The low-charge peaks had higher abundances and centered at 10+. The high-charge peaks had lower abundances and centered at 18+. Compared with the result obtained using 1% HAc, a higher charge state was observed here. Further increasing the concentration of NaCl to 5 mM resulted in a significant increase of the charge state (Figure 1b), as was discussed above. When the concentration of NaCl was increased to 10 mM, the charge state of Cyt c was slightly decreased (Figure S3b). A bimodal CSD was observed. The low-charge peaks appeared again, though with rather low abundances. They centered at 10+. The high-charge peaks had much higher abundances and centered at 17+.

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According to the results above, the concentration of NaCl was of great importance to the supercharging effect. Among the investigated concentrations, 5 mM NaCl gave the optimized result for 15 μM Cyt c samples. The obtained charge state was significantly higher than that obtained by 1% HAc. Supercharging of Cyt c by Other Chloride Salts. Besides NaCl, other chloride salts could also induce supercharging of proteins. Different chloride salts were investigated, including ammonium chloride (NH4Cl), potassium chloride (KCl) and lithium chloride (LiCl).

Figure 2. Supercharging of Cyt c by 5 mM NH4Cl (a) and 5 mM KCl (b). Cyt c was dissolved in ultrapure water in a concentration of 15 μM in both samples. PR-nESI was used for the detection of the samples.

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Supercharging of Cyt c by Other Sodium Salts. Sodium salts with different anions were investigated to see if the change of the anions would have an impact on the supercharging effect. The investigated salts included sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na 2HPO4), sodium iodide (NaI) and sodium nitrate (NaNO 3) (Figure 3). All the investigated sodium salts were capable of supercharging Cyt c. The results obtained under 5 mM NaH2PO4 and 5 mM Na2HPO4 were quite similar. Supercharging of Cyt c was observed under both salts. The CSDs ranged from 13+ to 21+ and centered at 17+. The qav was 16.9+ under NaH 2PO4 and 16.7+ under Na2HPO4. The presence of 2 mM NaI in sample also induced supercharging of Cyt c. The CSD ranged from 13+ to 21+ and centered at 18+. The qav was 17.2+. Supercharging of Cyt c was also achieved with 5 mM NaNO 3. A bimodal CSD was observed. The high-charge distribution had a much higher abundance, while the low-charge distribution had a lower abundance.

The result obtained under 5 mM NH4Cl (Figure 2a) was similar to that obtained under 5 mM NaCl. A unimodal CSD was observed. It ranged from 8+ to 22+ and centered at 18+. The qav was 16.6+. Supercharging of Cyt c was also achieved under 5 mM KCl (Figure 2b). The CSD ranged from 8+ to 21+ and centered at 17+. The qav was 16.2+. The use of LiCl still realized supercharging of Cyt c (Figure S4). The obtained CSD ranged from 8+ to 22+ and centered at 17+. The qav was 16.2+. According to the results above, four different kinds of chloride salts had been investigated, including LiCl, NaCl, KCl and NH4Cl. The change of the salt cations had little impact on the supercharging effect. All the investigated chloride salts were capable of supercharging Cyt c.

Figure 4. Detection results of Cyt c obtained under salts with no supercharging effect. Six salts were investigated, including NaHCO3 (a), NH4HCO3 (b), NaHS (c), (NH4)2S (d), NaAc (e) and NH4Ac (f). The concentrations of the salts were all 5 mM. Cyt c was dissolved in ultrapure water in a concentration of 15 μM in all the samples. PRnESI was used for the detection of the samples.

Figure 3. Supercharging of Cyt c by 5 mM NaH2PO4 (a), 5 mM Na2HPO4 (b), 2 mM NaI (c) and 5 mM NaNO3 (d). Cyt c was dissolved in ultrapure water in a concentration of 15 μM in all the samples. PR-nESI was used for the detection of the samples.

Salts with No Supercharging Effect. Not all salts were capable of supercharging Cyt c, such as carbonates and sulfides. Two bicarbonates were investigated, including sodium bicarbonate (NaHCO3) and NH4HCO3 (Figure 4a and b). No supercharging was observed under 5 mM NaHCO3. A unimodal CSD was observed. It ranged from 7+ to 13+ and centered at 9+. The qav was only 9.6+. Similar result was obtained under 5 mM NH4HCO3. The CSD ranged from 7+ to 15+ and cen-

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tered at 10+. The qav was 10.1+. In addition, ammonium carbonate ((NH4)2CO3) was also investigated (Figure S5). Still, no supercharging effect was observed. The CSD ranged from 7+ to 15+ and centered at 9+. The qav was 9.6+. Similar to carbonates, no supercharging effect was observed on sulfides (Figure 4c and d). Two sulfides were investigated, namely sodium bisulfide (NaHS) and ammonium sulfide ((NH4)2S). When NaHS was used, the CSD ranged from 7+ to 12+ and centered at 9+. The qav was 9.0+. When (NH4)2S was used, the obtained charge state was even lower. The CSD ranged from 7+ to 12+ and centered at 8+. The qav was 8.0+. Besides inorganic salts, organic salts were also investigated. Two acetates were investigated, including sodium acetate (NaAc) and NH4Ac (Figure 4e and f). The results of the two salts were quite similar. No supercharging was observed under both salts. The CSDs ranged from 7+ to 10+ and centered at 8+. The qav was 8.0+ under NaAc and 7.8+ under NH4Ac. In addition, ammonium formate (HCOONH4) was also investigated (Figure S6). The result was quite similar to that of the two acetates. The CSD ranged from 7+ to 11+ and centered at 8+. The qav was 8.0+. Observation of Anion Adducts. Based on the results above, it could be inferred that the supercharging effect of salts was related to their anion species. The anions interacted with protein molecules during the ionization process, resulting in the denaturation of protein structures.

Figure 5. Magnified spectra of Cyt c obtained under different salts, including 5 mM KCl (a), 5 mM NH4Cl (b), 5 mM NaH2PO4 (c), 5 mM Na2HPO4 (d), 2 mM NaI (e) and 2 mM LiI (f). Cyt c was dissolved in ultrapure water in a concentration of 15 μM in all the samples. PR-nESI was used for the detection of the samples.

In conventional nano-ESI, protein molecules formed metal cation adducts very easily. Figure S7 showed a representative nano-ESI mass spectrum of Cyt c. The sample was added

with 5 mM KCl. The spectrum was dominated by KCl clusters. No supercharging effect was observed. Only 8+ and 7+ charged Cyt c peaks were identified. The most abundant 8+ charged peak represented [M+7H+K] 8+ adduct. The most abundant 7+ charged peak represented [M+6H+K] 7+ adduct. Adducts with more than one K+ were also observed. No Cl− adduct was observed. When PR-nESI was carried out on the same sample, the result was totally different (Figure 2b). Figure 5a showed a magnified spectrum. According to the spectrum, KCl clusters and K+ adducts were completely removed. No KCl cluster or K+ adduct was identified. This was due to the highly efficient desalting effect of PR-nESI, which had been discussed in detail in our previous work. 46 However, Cl− adducts were identified in the spectrum. The formation of Cl− adducts indicated that Cl− and Cyt c molecules had considerable interactions. The interactions were strong enough to change the structure of Cyt c molecules, resulting in a denatured structure of Cyt c molecules. Subsequent ionization process generated the supercharged ions of Cyt c. The whole cycle of PR-nESI was composed of two steps, namely the negative high voltage step and the positive high voltage step. During the first step, a negative high voltage was applied to the sample (Figure 6a). The positively charged cations were driven away from the tip, while the negatively charged anions were driven towards the tip. The anions accumulated at the tip zone, resulting in a high concentration. For positively charged proteins, such as basic proteins, they were driven away from the tip. However, the protein molecules had huge volumes. They could only migrate a very limited distance.

Figure 6. The mechanism of PR-nESI for basic proteins. (a) The migration of different components in the sample during the negative high voltage step. (b) The migration of different components in the sample during the positive high voltage step.

The mass spectra obtained during the application of the negative high voltage were investigated. A typical spectrum of a Cyt c sample was shown in Figure S8a. The concentration of Cyt c was 15 μM. 5 mM NaCl was added to the sample. According to the spectrum, a rather weak signal was obtained and no signal of Cyt c was observed. In comparison, a much stronger signal was obtained when conventional nano-ESI was carried out with a negative high voltage of

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−1.5 kV (Figure S8b) for the detection of the same sample. The spectrum was dominated by NaCl clusters due to the presence of NaCl in the sample. No signal of Cyt c was observed. The results indicated that the negative high voltage of −3.0 kV in PR-nESI was too high to form a stable electrospray. A discharge process happened during this time, resulting in a decayed signal. When the positive high voltage step of PR-nESI came, the migration direction of the components reversed (Figure 6b). Protein molecules migrated towards the tip, while the anions migrated away from the tip. The two components met and interacted with each other, resulting in a changed structure of protein molecules. The solution flowed towards the tip, due to the electrospray at the tip. The denatured protein molecules were driven by the electric field and the flow of the solution towards the tip. Subsequent electrospray thus generated the supercharged ions of protein molecules. Cyt c, holoMb and Lys were basic proteins. They were positively charged in NaCl solutions. They behaved according to Figure 6 during the supercharging process. For negatively charged proteins, such as acidic proteins, the result was similar (Figure 7). During the negative voltage step, cations were driven away from the tip. On the contrary, protein molecules and anions were driven towards the tip. Protein molecules and anions accumulated at the tip zone. They interacted with each other, resulting in a denatured structure of protein molecules. When the positive voltage step came, protein molecules were directly sprayed out from the tip. CaM was an acidic protein. It was negatively charged in NaCl solution. It behaved according to Figure 7 during the supercharging process.

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These studies showed that the existence of salt ions in solution could have strong enough impact on the structure of protein molecules. In this work, the adducts of proteins and salt anions were observed, indicating an interaction between them. In addition to KCl, other salts were also investigated, including NH4Cl, NaH2PO4, Na2HPO4, NaI and LiI. NH4Cl was another chloride salt. Cl− adducts were also identified in the spectrum (Figure 5b). NaH2PO4 and Na2HPO4 were phosphates. H2PO4− adducts were identified in their spectra (Figure 5c and d). Adducts with one, two, three and even more H2PO4− anions were observed. NaI and LiI were iodide salts. I− adducts were identified in their spectra (Figure 5e and f). Adducts with different numbers of I − anions were observed. There observations further confirmed that Cyt c molecules had considerable interactions with the anions of these salts during PR-nESI. Cyt c was a basic protein. To further confirmed the interactions between anions and acidic protein molecules, the adducts of CaM and anions were investigated. As was shown in Figure S9, anion adducts of CaM could be observed in the magnified mass spectra. NaH2PO4 and LiI were used for the supercharging of CaM. When NaH2PO4 was used for the supercharging of CaM, adducts with different numbers of H2PO4– anions were observed (Figure S9a). Similar result was obtained on LiI (Figure S9b). When LiI was used for the supercharging of CaM, adducts with one, two and three I– anions were observed. According to the mechanism proposed above, the desalting process was actually driven through an electrophoresis mechanism. Solution heating would occur during the process. However, its impact on protein structure was very limited during PR-nESI. When positive polarity was used for the detection of proteins, the negative high voltage of −3.0 kV was applied to the solution for only 5−10 seconds. Then the polarity of the high voltage was reversed and mass spectra were obtained. The time was too short for the accumulation of heat. The increase of the solution temperature was very limited. Thus the structure of proteins would hardly be changed. Furthermore, no unfolding of Cyt c was observed during PRnESI when weak acid salts were added to the samples, such as NaAc and NH4Ac (Figure 4e and f). When no additive was added to the sample, the CSD of Cyt c obtained by PR-nESI was quite similar to that obtained with NaAc and NH4Ac (Figure S1c). The structure of Cyt c remained folded during PR-nESI no matter if the samples were added with salts or not. This indicated that the addition of salts to the samples would not cause significant solution heating during PR-nESI. The CSD of Cyt c obtained by nanoESI with no additive in sample (Figure 1a) was similar to that obtained by PR-nESI. A folded structure of Cyt c was observed. This further confirmed that the electrophoresis process in PRnESI had little impact on the structure of proteins whether with or without salts in samples. Detection of proteins in negative polarity. Proteins could also be detected in negative polarity by PR-nESI. Cyt c was dissolved in ultrapure water in a concentration of 15 μM. NaCl was added to the solution in a concentration of 5 mM. The obtained sample was detected by both nano-ESI and PRnESI. When nano-ESI was used for the detection of the sam-

Figure 7. The mechanism of PR-nESI for acidic proteins. (a) The migration of different components in the sample during the negative high voltage step. (b) The migration of different components in the sample during the positive high voltage step.

Studies have shown that buffer ions could affect the stability, solubility and function of proteins through nonspecific interactions.47-49 Additives in solutions could cause unfolding or conformational changes of proteins, presumably by displacement and/or interference with critical electrostatic interactions.50 It has also been proved that the concentration of buffers could have considerable influence on non-covalent interactions between receptor−ligand binding affinities.51

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ple, the spectrum was dominated by NaCl clusters (Figure S10a). No signal of Cyt c could be identified. When PR-nESI was used for the detection of the sample, a unimodal CSD of Cyt c was observed (Figure S10b). It ranged from 8− to 12− and centered at 10−. Cation adducts were observed (Figure S10c). Adducts with one, two, three and even more Na + were identified from the amplified spectrum of the 10− charged Cyt c ions. The positive and negative polarities of PR-nESI followed opposite mechanisms. When positive polarity was used for the detection, proteins formed anion adducts even if they were acidic proteins (such as CaM). When negative polarity was used for the detection, proteins formed cation adducts even if they were basic proteins (such as Cyt c). One protein could be detected in both positive and negative polarities. Anion adducts of the protein would be observed in positive polarity, while cation adducts of the protein would be observed in negative polarity. The signal intensity and signal-to-noise ratio (S/N). The signal intensity and S/N of Cyt c obtained with different salts in samples were calculated and compared, as was shown in Figure S11a and b. Cyt c was dissolved in ultrapure water in a concentration of 15 μM. The obtained solution was detected by nano-ESI. Then, aqueous solutions of Cyt c with different kinds of salts were prepared and detected by PR-nESI. The investigated salts included NaCl, NaNO 3, sodium formate (HCOONa) and NaAc. The concentrations of the salts were all 5 mM. The signal intensities of Cyt c were shown in Figure S11a. For each mass spectrum, the signal intensity of the highest peak of Cyt c was counted. The charge state of the highest peak of Cyt c obtained with different salts was shown in Figure S11c. the When no salt was added to the sample, a quite weak signal was obtained by nano-ESI. When the two strong acid salts (NaCl and NaNO3) were added to the samples, much higher signal intensities were obtained by PR-nESI. NaCl gave the highest signal intensity. When the two weak acid salts (HCOONa and NaAc) were added to the samples, the signal intensities were much lower than that obtained with the two strong acid salts. They were only a little higher than that obtained by nano-ESI with no salt. NaAc gave the weakest signal intensity among all the salts. Quite similar results were obtained on S/N, as was shown in Figure S11b. For each mass spectrum, the signal intensities of the noises within the mass range of m/z = 1050−1100 were averaged to give a mean noise. The signal intensity of the highest peak of Cyt c was compared with the mean noise to give the S/N. When no salt was added to the sample, a low S/N was obtained by nanoESI. When the two strong acid salts were added to the samples, much higher S/N were obtained by PR-nESI. NaCl gave the best S/N. When the two weak acid salts were added to the samples, the S/N were much lower than that obtained with the two strong acid salts. The S/N obtained with HCOONa was slightly higher than that obtained by nano-ESI with no salt. The S/N obtained with NaAc was almost the same with that obtained by nano-ESI with no salt. In conclusion, when different salts were added to the samples, the signal intensity and S/N obtained by PR-nESI were dependent on the species of the salts. The strong acid salts

gave much higher signal intensities and S/N, while the weak acid salts gave much lower signal intensities and S/N. Compared with the result obtained by nano-ESI with no salt in the sample, our method did not cause a decrease of the signal intensity and S/N even though high concentrations of salts were added to the samples. The Difference Between Strong Acid Anions and Weak Acid Anions. Among the investigated anions, those strong acid anions were capable of supercharging proteins. For example, Cl−, Br− (Figure S12), I− and NO3− had been proven to have supercharging effect. H 3PO4 was a medium-strong acid. Its conjugated anions, namely H2PO4− and HPO42−, also had supercharging effect. In comparison, those weak acid anions were not able to supercharge protein molecules. For example, HCO3−, HS−, Ac− and HCOO− showed no supercharging effect. The key difference between strong acid anions and weak acid anions was that weak acid anions hydrolyzed severely in aqueous solution. The equilibrium concentration of weak acid anions in solution was significantly lower than the analytical concentration value, due to hydrolization. As was shown in Figure S3, the supercharging effect was dependent on the concentration of the anion. The effect decayed rapidly with the decrease of the anion concentration. The severe hydrolysis remarkably decreased the actual concentration of weak acid anions. The interaction between protein molecules and weak acid anions was then reduced to a quite limited extent. Thus, the structure of protein molecules could hardly be changed by the weak acid anions. The pH of the solution would change during the ESI process, which had previously been describe in detail in literatures.52-56 In positive polarity, the pH decreased due to the oxidation reactions within the nano-tip, such as 2H2O → 4H+ + 4e− + O2.52 For unbuffered solutions, the pH could decrease from near neutral to pH = 3.53 In addition, the decrease of pH could also take place within the charged droplets generated by ESI.54,55 This was due to the evaporation of the solvent and the enrichment of H+ within the droplets.1,2 The effective pH of the droplets near the moment of ion formation could be 1−3 pH units lower than the pH of the solution prior to ESI.57,58 The influence of solvent pH and protein pI on the ionization responses of proteins had previously been studied.10 Mass spectra of proteins obtained by nano-ESI was dependent on the pH of the solution. When positive polarity was used for the detection of proteins, the maximum signals were obtained when the pH value of the solution was 4−5 units lower than the protein pI. Na+ adducts were minimized at the same time. Na+ adducts with higher abundances were observed at higher pH values. Decayed signals were obtained at the same time. Mass spectra of CaM obtained by nano-ESI with different solution pH were shown in Figure 1e, Figure S2b and Figure S13a. When no additive was added to the solution, the solution had a near-neutral pH. A weak signal of CaM was observed with severe metal ion adducts (Figure 1e). The addition of 667 μM HAc to the solution decreased the pH to 4.0. Taking into consideration of the pH decrease during ESI, the effective pH of the spray droplets would be lower than 4.0. The signal of CaM was improved a little, but still quite weak (Figure S13a). Further increasing the concen-

7



tration of HAc to 56.8 mM resulted in a decrease of pH down to 3.0. The effective pH of the spray droplets would be lower than 3.0. The signal of CaM was significantly enhanced (Figure S2b). No metal ion adduct was observed. PR-nESI was capable of ionizing acidic proteins, which had previously been demonstrated in our work.46 A representative mass spectrum of CaM obtained by PR-nESI was shown in Figure S13b. No additive was added to the solution. A strong signal of CaM was observed with no metal ion adduct. The signal was much stronger than that obtained by nano-ESI with a solution pH of 4.0. It was comparable to that obtained by nano-ESI with a solution pH of 3.0. Based on the results above, we conjecture that the effective pH of the spray droplets generated by PR-nESI was within the range of 2−3, or even lower. At such a pH value, severe hydrolization of weak acid anions would occur. Imitation of the supercharging effect of PR-nESI in conventional nano-ESI. According to the results above, the key that led to the supercharging effect of PR-nESI included two factors. One factor was a solution pH lower than 3.0. The other factor was the presence of salt anions in solution. The supercharging effect of PR-nESI was caused by both the binding of anions and the influence of H+. To test this conclusion, Cyt c solutions with certain pH and salts were prepared and detected by nano-ESI (Figure S14). A representative mass spectrum of Cyt c obtained by nanoESI with the presence of 1% HAc in solution was shown in Figure S1a. The presence of 1% HAc in solution offered a pH of 2.8. A bimodal CSD was observed. The low-charge peaks had much higher abundances and centered at 9+, while the high-charge peaks had lower abundances and centered at 17+. When 1 mM NaCl was added to the solution together with 1% HAc, the charge state of Cyt c was significantly increased (Figure S14a). The addition of a bimodal CSD was also observed. The high-charge peaks had higher abundances and centered at 16+, while the low-charge peaks had lower abundances and centered at 8+. Further increasing the concentration of NaCl to 5 mM resulted in an even higher charge state of Cyt c (Figure S14b). The abundances of the low-charge peaks were further reduced to a lower level. When 5 mM NaAc was added to the solution together with 1% HAc, a bimodal CSD was observed (Figure S14c). The lowcharge peaks had higher abundances and centered at 9+, while the high-charge peaks had lower abundances and centered at 14+. The overall qav of Cyt c was 11.1+. In comparison, the overall qav of Cyt c obtained without NaAc was 10.6+ (Figure S1a). The overall charge state of Cyt c obtained with NaAc was slightly higher than that obtained with no salt. According to the results above, the addition of NaCl in solution remarkably increased the charge state of Cyt c. The charge state of Cyt c was even higher when the concentration of NaCl was increased from 1 mM to 5 mM. In comparison, the charge state of Cyt c was only increased to a very limited extent when 5 mM NaAc was added to the solution. The cation of the two salts was the same. Thus the difference of the charge state obtained under the two salts should be due to their anions. HAc was a quite weak acid. Severe hydrolization of Ac− would occur in aqueous solution, especially when the solution had a low pH. Thus the actual concentration of Ac− would be reduced to a very low level. In comparison, HCl was a strong acid. Cl− would not hydrolyze in

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aqueous solution. Thus the actual concentration of Cl− in solution would not be affected by hydrolization. As Cl− had a much higher actual concentration than Ac−, it would have a stronger impact on the structure of Cyt c. Studies have shown that buffer ions could affect the stability, solubility and function of proteins through nonspecific interactions.47-49 The effects of various salts on protein structure and stability have been investigated extensively.59-61 The presence of certain salt ions in solution enhances protein denaturation. Anions tend to have more significant contribute to the conformational changes of proteins than cations.62 The binding of anions to protein molecules could affect the stability of protein molecules. For example, the binding of phosphate ions to Cyt c molecules has been proved to destabilize Cyt c molecules and increase the flexibility of the heme region.63,64 In PR-nESI, the binding of strong acid anions to protein molecules were observed on both basic and acidic proteins (Figure 5 and Figure S9). Considerable interactions between the anions and protein molecules happened. Furthermore, a solution pH of about 2−3 was created within the spray droplets during PR-nESI. The combination of strong acid anions and H+ resulted in the denaturation of proteins. Supercharging of the protein molecules was thus achieved. However, when weak acid salts were added to the samples, no supercharging was observed due to the hydrolization of weak acid anions. Although the supercharging effect of PR-nESI could be repeated on nano-ESI, the use of PR-nESI was simpler from an experimental perspective. No acid was needed in the solution. In addition, the realization of the supercharging effect on nano-ESI further confirmed our conclusion that the pH of the solution and the anions in solution played vital roles during the supercharging process. Unlike PR-nESI, the pH of the spray droplets generated by conventional nano-ESI was not low enough. Thus no supercharging was observed even if strong acid salt was added to the sample. Additional acid was needed. When both strong acid salt and acid were added to the sample, the supercharging effect of PR-nESI could successfully be repeated on conventional nano-ESI. Applications to the analysis of biological samples. To further confirm the capability of our method for the analysis of actual biological samples, our method was applied to the detection of a Cyt c sample extracted from pig heart. Natural extraction was one of the most commonly used methods for the preparation of proteins. It was widely used in biological researches. Generally, proteins were extracted from animal or plant tissues. In this study, Cyt c was extracted from pig heart (Detailed description of the extraction procedure was shown in the Supporting Information). After extraction, the sample was kept in 1×PBS. PBS was a widely used biological buffer. It offered a stable neutral pH, which was advantageous to the maintenance of native protein structures. The concentration of Cyt c in the PBS was 5 mg/mL, which was measured by a NanoDrop Spectrophotometer. Before analysis, the sample was diluted with ultrapure water by 30 times. The obtained solution was directly detected by nanoESI and PR-nESI. When nano-ESI was used for the detection of the sample, no signal of Cyt c was observed (Figure S15a). NanoESI failed in the detection of the Cyt c sample due to the presence of PBS and other impurities in the sample. In comparison, when

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PR-nESI was used, Cyt c was successfully detected (Figure S15b). A unimodal charge state distribution (CSD) of Cyt c was observed. The CSD ranged from 10+ to 20+ and centered at 16+. The qav of Cyt c was 16.1+. The commercial 1×PBS contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM potassium dihydrogen phosphate (KH2PO4). The overall concentration of Cl− was 140 mM. The overall concentration of H2PO4− and HPO42− was 9.6 mM. When the sample was diluted by 30 times, the final concentrations of Cl− and H2PO4−/HPO42− were 4.67 mM and 0.32 mM, respectively. The concentrations of the salts were just suitable for the supercharging of proteins by PR-nESI. When detecting proteins in biological samples, the difficulty lay in the presence of the biological buffer used in the solutions. To further confirm the capability of our method for the detection of proteins in PBS, a standard solution of holo-Mb was investigated. Holo-Mb was dissolved in 1×PBS in a concentration of 300 μM. Before analysis, the sample was diluted with ultrapure water by 30 times. The obtained solution was directly detected by nanoESI and PR-nESI. When nano-ESI was used for the detection of the sample, the obtained mass spectrum was dominated by salt clusters (Figure S16a). No signal of Mb was observed. In comparison, when PR-nESI was used for the detection of the sample, a unimodal CSD of apo-Mb was observed (Figure S16b). The CSD ranged from 12+ to 27+ and centered at 22+. The qav of apo-Mb was 21.0+. In addition to PBS, another commonly used biological buffer was also investigated, namely the Tris-HCl buffer. Cyt c was dissolved in 50 mM Tris-HCl buffer (pH = 7.4) in a concentration of 120 μM. Before analysis, the sample was diluted with ultrapure water by 8 times. The obtained solution was directly detected by nano-ESI and PR-nESI. No signal of Cyt c was observed when nano-ESI was used for the detection of the sample (Figure S17a). The spectrum was dominated by Tris-HCl clusters. In comparison, a unimodal CSD of Cyt c was observed when PR-nESI was used for the detection of the sample (Figure S17b). The CSD ranged from 9+ to 21+ and centered at 16+. The qav of Cyt c was 15.8+. The 50 mM Tris-HCl buffer used here contained 50 mM tris(hydroxymethyl)aminomethane (Tris) and 42 mM HCl. The concentration of Cl− in the buffer was 42 mM. When the sample was diluted by 8 times, the final concentration of Cl− was 5.3 mM in the sample. The concentration of Cl− was just suitable for the supercharging of proteins by PR-nESI. Mb was also investigated to confirm the capability of our method for the detection of proteins in Tris-HCl buffer. Holo-Mb was dissolved in 50 mM Tris-HCl buffer (pH = 7.4) in a concentration of 80 μM. Before analysis, the sample was diluted with ultrapure water by 8 times. The obtained solution was directly detected by nano-ESI and PR-nESI. No signal of Mb was observed in the mass spectrum obtained by nano-ESI (Figure S17c). The spectrum was dominated by Tris-HCl clusters. In comparison, Mb was successfully detected by PR-nESI. A unimodal CSD of apo-Mb was observed in the obtained mass spectrum (Figure S17d). The CSD ranged from 12+ to 27+ and centered at 22+. The qav of apo-Mb was 20.9+. Many biological samples are buffered with salts. The analysis of such samples by conventional nano-ESI generally requires pretreatments, such as dialysis or ultrafiltration. These pre-treatments

are time consuming and require special instruments. Furthermore, additional supercharging reagents are needed in the solution to achieve supercharging of proteins. In comparison, with our method, such biological sample can be analyzed directly. Supercharging of proteins can be achieved without any other additive or pretreatment. Our method is much more simple and practical from an experimental perspective.

CONCLUSIONS In this work, a novel method for the supercharging of proteins was developed. It made use of PR-nESI with the presence of 1−10 mM salt in solution to achieve supercharging of proteins. High concentrations of salt anions were accumulated at the tip zone during the negative high voltage step of PR-nESI. The anions interacted with protein molecules, resulting in a denatured structure of protein molecules. Subsequent ionization process during the positive high voltage step of PR-nESI generated the supercharged protein ions. The species of the anion was critical to the supercharging effect. Different salts were investigated. Those with strong acid anions were capable of supercharging proteins. The signal intensity and S/N of proteins could be increased at the same time. In comparison, salts with weak acid anions had no supercharging effect. The present work offered us an alternative way for the supercharging of proteins. The use of common salts for supercharging made it more convenient. The concentration of salts needed for supercharging was much lower than those conventionally used supercharging reagents. Taking into consideration of the fact that many biological samples were buffered with phosphates and chlorides, these samples could directly be supercharged by our method without any additional additives. Furthermore, many salts were non-toxic and could easily be found in a chemical lab, the use of salts for supercharging would be a much more practical and economic choice. In addition, the present work also furthered our understandings about the mechanism of supercharging, as well as electrospray.

ASSOCIATED CONTENT Supporting Information. Additional mass spectra referred to in the manuscript, including: experimental details; the detection results of Cyt c by PR-nESI and nano-ESI with different additives in solution; the detection results of CaM by PR-nESI and nano-ESI with different additives in solution; the signal intensity and S/N of Cyt c obtained by PR-nESI with different salts in samples; the detection results of Cyt c sample extracted from pig heart by nano-ESI and PR-nESI; the detection results of holo-Mb dissolved in PBS by nano-ESI and PR-nESI; the detection results of Cyt c and holo-Mb dissolved in Tris-HCl buffer by nano-ESI and PR-nESI.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

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Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Nos. 21605135 and 21575132) and the Ministry of Science and Technology of China (No. 2016YFF0102603). We sincerely thank our teammates for their help in the experiments, including Jiafeng Song, Shiying Chu and Guangcai Liu. We also thank Yuxuan Luo for his help in literatures.

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Supercharging of different proteins by NaCl, including Cyt c, holo-Mb, CaM and Lys. Proteins were dissolved in ultrapure water. The concentrations of Cyt c, holo-Mb, CaM and Lys were 15 μM, 10 μM, 5 μM and 20 μM, respectively. Representative nano-ESI mass spectra of Cyt c, holo-Mb, CaM and Lys were shown in (a), (c), (e) and (g), respectively. Proteins were dissolved in ultrapure water. The obtained solutions were directly detected by nano-ESI. The detection results of Cyt c, holo-Mb, CaM and Lys obtained by PR-nESI with the presence of 5 mM NaCl in samples were shown in (b), (d), (f) and (h), respectively. Proteins were dissolved in ultrapure water. 5 mM NaCl was added to the solutions. The obtained solutions were detected by PR-nESI. 199x282mm (300 x 300 DPI)



Supercharging of Cyt c by 5 mM NaH2PO4 (a), 5 mM Na2HPO4 (b), 2 mM NaI (c) and 5 mM NaNO3 (d). Cyt c was dissolved in ultrapure water in a concentration of 15 μM in all the samples. PR-nESI was used for the detection of the samples.


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Magnified spectra of Cyt c obtained under different salts, including 5 mM KCl (a), 5 mM NH4Cl (b), 5 mM NaH2PO4 (c), 5 mM Na2HPO4 (d), 2 mM NaI (e) and 2 mM LiI (f). Cyt c was dissolved in ultrapure water in a concentration of 15 μM in all the samples. PR-nESI was used for the detection of the samples. 209x227mm (300 x 300 DPI)


Supercharging of Proteins by Salts during Polarity Reversed Nano

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