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Nanoemitters Suppress the Formation of Metal Adduct Ions in Electrospray Ionization Mass Spectrometry Jun Hu, Qi-Yuan Guan, Jiang Wang, Xiao-Xiao Jiang, ZengQiang Wu, Xing-Hua Xia, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04218 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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Analytical Chemistry

Nanoemitters Suppress the Formation of Metal Adduct Ions in Electrospray Ionization Mass Spectrometry Jun Hu, Qi-Yuan Guan, Jiang Wang, Xiao-Xiao Jiang, Zeng-Qiang Wu, Xing-Hua Xia, Jing-Juan Xu,* Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China * Corresponding author. Tel/Fax: +86-25-89687294; E-mail address: [email protected]

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ABSTRACT In the work, we showed that the use of nanoemitters (tip dimension < 1 micron, typically ~100 nm) could dramatically reduce the nonspecific metal adduction to peptide or protein ions as well as improve the matrix tolerance of electrospray ionization mass spectrometry (ESI-MS). The proton-enriched smaller initial droplets are supposed to have played a significant role in suppressing the formation of metal adduct ions in nanoemitters. The proton enrichment effect in the nanoemitters is related to both the exclusion-enrichment effect (EEE) and the ion concentration polarization effect (ICP effect), which permit the molecular ions to be regulated to protonated ones. Smaller initial charged droplets generated from nanoemitters need less fission steps to release the gas phrase ions, thus, the enrichment effect of salt was not as significant as that of microemitters (tip dimension > 1 micron), resulting in the disappearing of salt cluster peaks in high m/z region. The use of nanoemitters demonstrates a novel method for tuning the distribution of the metal adducted ions to be in a controlled manner. This method is also characterized by ease of use and high efficiency in eliminating the formation of adduct ions and no pre-treatment such as desalting is needed even in the presence of salt at millimole concentration.

KEYWORDS: mass spectrometry, matrix interference, exclusion-enrichment effect, ion transport, nanoemitters, peptides, proteins.

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INTRODUCTION Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool to identify peptides and proteins.1,2 However, the performance of ESI-MS including nanoelectrospray methods (nESI) can be susceptible because of the formation of metal adduction, when some salts (even at trace amounts) are also present in the samples.3-5 Alkali salts, especially sodium salts, are ubiquitous in nature. The signal intensity of the peptides or proteins can be reduced by the signal spreading among the multiple sodiated analyte ions.6,7 The presence of salts in the samples can also complicate the mass spectra as well as depress the sensitivity of ESI-MS because of the ion suppression effect.5 Therefore, desalting is a universal practice in ESI-MS. Several strategies have been demonstrated to eliminate the formation of metal adduction in ESI-MS. One common approach is to remove the salt from the solution prior to ESI-MS, which can be carried out by coupling with several on-line or off-line separating techniques, such as liquid chromatography, capillary electrophoresis, ion exchange, solid phase extraction etc.8-10 However, these methods may suffer from the diffusion of analytes in the liquid phase, which may reduce the sensitivity. Using solution additives is another approach to regulate the adduct ions distribution.11-14 The addition of supercharging reagents, such as m-nitrobenzyl alcohol (m-NBA) or volatile salts, such as ammonium bromide (NH4Br) has been shown to reduce sodium adduction in ESI-MS. Although, the sodium adduction to peptides or proteins can be reduced significantly, the ion abundance of analyte of interest could be depressed by ion suppression caused by the additives. Ion/ion or ion/molecule reaction in the electrospray generated aerosol can also be used to regulate the formation of metal adduction.15,16 McLuckey's group has reported that exposing the electrospray droplet to 20% acetonitrile vapor can efficiently remove metal ions from proteins and protein complexes.15 However, it needs to introduce special reagents and devices for ion/ion or ion/molecule reactions, which complicate the experimental setup. Additionally, the process of these methods is relatively tedious and hard to be applied to the measurement of ultra-small volume samples such as single cells. Recently, electrospray ionization methods based on capacitive coupling were introduced by Cooks et al. An induced nanoelectrospray (induced nESI) method was developed, in which periodically pulsed high voltage was applied on an electrode within 2 mm away from nanospray emitters to induce discontinuous electrospray, and high-salt samples such as serum and urine could be analyzed directly with high sensitivity.17 In our previous study, we developed an induced nESI based method termed as synchronized polarization induced electrospray ionization (SPI-ESI) to simultaneously obtain both


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positive-ion and negative-ion mass spectra from single-cell samples,18 in which micrometers (tip dimension > 1 micron) were mainly used for single-cell sampling and electrospray ionization. Baker et al. have reported that glass pipettes pulled to orifice diameters of less than 100 nm can be used as robust electrospray ionization emitters for mass spectrometry,19 and they further employed the nano-sized pipettes for local sampling of single cells for subsequent matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis.20 With the decrease of the emitter tip dimension, surface area relative to the solution volume in the emitter tips increases significantly. Williams et al. have reported that strong interaction between the protein molecules and the negatively charged glass surface could occur in the nano-sized emitters, which led the unfolding of proteins with a net positive charge.21 On account of the high surface-to-volume ratio in nano-sized channels, many unique phenomena of liquids in confined geometries have already been reported, including the enrichment of cations (e.g. oxonium ions) and exclusion of anions induced by the negatively charged glass surface.22,23 Ion transport is an important process in ESI. Taking the positive-ion mode as an example, ion transport causes cations (oxonium ions dominated, due to the high mobility of oxonium ions) to migrate toward the tip of emitter, causing strong acidification of solution in the front zone of the emitter tips.24,25 By taking full advantage of the unique properties of solution inside the nanoemitters (tip dimension < 1 micron, typically ~100 nm, so called nanoemitters), we demonstrate here a novel method, which utilizes nanoemitters to suppress the formation of nonspecific metal adduction and salt cluster in ESI-MS. As was illustrated in Scheme 1, emitters were placed about 5 mm away from the traction electrode (MS inlet) and pulsed repeatedly high potential (at 0.5 Hz) was applied onto the traction electrode to generate electrostatic field for polarization induced electrospray ionization (PI-ESI).18 Typically, in nESI, inserting a metal wire into the emitters or coating the emitter with conductive material is indispensable to make efficient electrical contacts.26-28 Unlike that of nESI, PI-ESI method is characterized by ease of use, since no physical contact between the solution and electrode is needed. Besides the reduction of metal adduction to peptide or protein ions, the use of nanoemitters in PI-ESI also significantly improve the matrix tolerance of ESI-MS, no desalting process is needed even in the presence of salt at tens of millimole concentration. The goal of the present study is to demonstrate a new approach to suppress the nonspecific metal adduction to peptide or protein ions via utilizing nanoemitters with the objective to reveal the underlying mechanism.

EXPERIMENTAL SECTION


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Leu-enkephalin, angiotensinⅡ, cytochrome c, lysozyme (from hen egg white), formic acid (FA), NaCl, KCl, CaCl2, MgCl2, NH4Br, 18-Crown-6 (18C6), HPLC grade methanol, and acetonitrile were purchased from Sigma. Recombinant human insulin was purchased from Beijing Century Aoke Biotechnology. All samples were used without further purification. Deionized water (18.2 MΩ·cm) was obtained using a Milli-Q system. The borosilicate glass capillaries (i.d. = 0.58 mm, o.d. = 1 mm, with filament) were purchased from Sutter Instruments. Emitters with varying tip dimensions (Figure 1) were prepared using a laser puller (P-2000, Sutter Instrument) and characterized by a field emission scanning electron microscope (S-4800, Hitachi). The parameters settings of the laser puller can be found in the Supporting Information. All the solutions were kept in polypropylene centrifuge tube (Corning) before mass spectrometric analysis. Sample solution was loaded into the emitter up front using a microloader (Eppendorf). Mass spectra were acquired using a Q-TOF (6530B, Agilent) mass spectrometer with a data acquisition rate of 1 Hz. Except as noted, the instrument parameters were as follows: gas temp = 300 °C, drying gas = 2 L/min, nebulizer = 0 psig. Pulsed high potential (at 0.5 Hz) was obtained by adding two segments with cycle time = 2 s, one segment was set at 3.5 kV and the other segment was set at 0 kV.

RESULTS AND DISCUSSION Effects of Emitter Tip Dimension on Metal Ion Adduction To demonstrate the capability of nanoemitters in reducing the formation of metal ion adduction, emitters with varying aperture were used to evaluate the tuning efficiency. Na+ is ubiquitous in nature and is chosen as a model metal ion to evaluate the interaction with Leu-enkephalin. Mass spectra obtained with ~60 nm, ~120 nm, ~600 nm and ~1200 nm emitters were shown in Figure 2a-d, respectively. In the presence of 0.1 mM NaCl, almost no peaks corresponding to sodiated ions were observed when ~ 60 nm emitters were used (Figure 2a) and the sodiated adducts was only a small fraction of the total ions (3.5%, m/z range: 540-630) when increase the emitter tip dimension to ~120 nm (Figure 2b). However, mono-sodiated ions dramatically become the most abundant ion species when further increase the emitter tip dimension to ~600 nm (Figure 2c). In the spectra obtained with ~1200 nm emitters (Figure 2d), the ratio of di-sodiated ions increase further and protonated ions disappeared. The consistent shifting trend to multiple sodiated ion form as the increase of emitter tip dimension indicated that the composition of adduct ions could be accurately tuned using emitters with appropriate aperture, which usually requires changing the conditions of the spray solvent such as pH or desalting


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carefully.29,30

Matrix Tolerance of Nanoemitters The elimination of metal adduction to Leu-enkephalin ions in nanoemitters made us infer that nanoemitters can achieve higher matrix tolerance in comparison with microemitters. Thus, we test the matrix tolerance of microemitters (~1.2 µm) and nanoemitters (~120 nm). Firstly, the effects of salt concentration on metal ion adduction were investigated with Leu-enkephalin using ~1.2 µm microemitters and NaCl with different concentration was added into the aqueous solution as an artificial matrix. The results summarized in Figure 2d and upper panel of Figure 3 showed the positive-ion mass spectra obtained with ~1.2 µm microemitters from 10 µM Leu-enkephalin aqueous solutions containing 0.1, 1, 10 and 25 mM NaCl (the analyte/salt ratio decreased from 1/10 to 1/2500). The addition of 1 mM NaCl into the aqueous solution of 10 µM Leu-enkephalin (Figure 3a) resulted in the formation of salt cluster ions and molecular ions with multiple sodium ion adduction. When further increase the salt concentration to 10 mM (Figure 3b), peaks from salt cluster ions were dominant and their intensities were several times higher than that of the di-sodiated Leu-enkephalin ions [M+2Na-H]+. The concentration of added NaCl in Figure 3b was only ten times of that in Figure 3a, but the abundance of Leu-enkephalin ions decreased more than 30 times. Due to the severe ion suppression effect caused by NaCl, Leu-enkephalin was failed to be detected when the concentration of NaCl was larger than 25 mM (figure 3c). Mass spectra obtained with nanoemitters from 10 µM Leu-enkephalin aqueous solutions containing different concentration of NaCl were shown in the lower panel of Figure 3. In comparison with the results from microemitters, a substantial reduction in the formation of sodium ion adduction was observed (e.g. Figure 3a vs. Figure 3d). In spectra obtained with microemitters (Figure 3a), di-sodiated ions [M+2Na-H]+ was the most abundant molecular ion species and nearly no protonated ions was observed. The average number of sodium ions adducted to Leu-enkephalin was 1.61±0.04. While in Figure 3d (obtained with ~120 nm nanoemitters from the same solution used in Figure 3a), protonated ions was the most abundant and the average number of sodium ions adducted to Leu-enkephalin was reduced to 0.25±0.01. A great number of peaks corresponding to salt cluster (such as [Na10Cl9]+ and [Na11Cl10]+, shown in Figure 3a) were also introduced into the mass spectra in microemitters, but the spectrum was much more ''clean'' in nanoemitters (Figure 3d). More significant matrix tolerance of nanoemitters was observed when the salt concentration was increased to 10 mM. As was shown in


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Figure 3b, Leu-enkephalin was detected mainly as di-sodiated ions [M+2Na-H]+ with a signal-to-noise ratio (SNR) of 29.5, but almost be submerged in the salt cluster dominated peaks. Whereas in nanoemitters (Figure 3e), the most abundant peaks in the spectrum was from mono-sodiated ions [M+Na]+, and the SNR was 280.2, indicating about one order of magnitude higher matrix tolerance towards salt contamination than that of microemitters. This meant that, in order to give comparable mass spectra, microemitters require about ten times lower NaCl concentration (see Figure 3a vs. Figure 3e). The high matrix tolerance towards salt contamination make it possible to distinguish Leu-enkephalin signals even at 25 mM NaCl concentration (Figure 3f, the SNR was 9.1 for [M+2Na-H]+). From the preliminary results, it was clear that the increase of salt/analyte ratio would reduce the signal intensity of the analyte ions and regulate the analyte ions to be multiple sodiated ones, but a better tolerance against the matrix can be achieved by using nanoemitters.

Universality of Nanoemitters on Reducing the Nonspecific Metal Adduction to Peptide or Protein Ions To further demonstrate that the use of nanoemitters can be a universal approach to suppress the nonspecific metal adduction to peptide or protein ions, other compounds including angiotensin Ⅱ and insulin were also analyzed using ~120 nm nanoemitters and ~1.2 µm microemitters. As was shown in Figure 4, it was very obvious that the peaks with same charge state were extended by the multiple sodiated adducts in spectra obtained with microemitters (Figure 4a and Figure 4c, for angiotensin Ⅱ and insulin, respectively). However, in mass spectra obtained with nanoemitters, the most abundant ion species were regulated to be protonated ones (see Figure 4b and Figure 4d). Taking angiotensin Ⅱ for example, the molecular ion abundance was distributed by the multiple sodiated adducts in microemitters and the number of sodium ion in the adducts can be extended up to 4 in peaks of charge state of 2+ (Figure 4a). More noticeable multiple sodiated adducts were observed in insulin and up to 12 sodium ions can be attached to the insulin ions of charge state of 5+ (Figure 4c). However, in spectra obtained with nanoemitters, mono-sodiated adduct was the only observed adduct form and its proportion was very low (see, Figure 4b and Figure 4d, for angiotensin Ⅱ and insulin, respectively). It was interesting that the ion abundance from nanoemitters were several times higher than that of microemitters. No peak broadening in nanoemitters may partly contribute to this result. The smaller orifice of nanoemitters used here can decrease the initial droplet diameters and increase the charge-to-surface area ratios of the initial droplets, which is very advantageous for the formation of gas-phase ions.3 Therefore, the ion abundance


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may also be attributable, in part, to the higher ionization efficiency from the smaller initial charged droplets in nanoemitters. Another observation was the shifting of the charge state distribution to a higher average charge state (peaks shifted to lower m/z region) in nanoemitters, which was in good agreement with the previous reports.19 Taking insulin as an example, an average charge state of 4.3+ was observed in microemitters (Figure 4c). However, the average charge state shifted to 6.1+ in nanoemitters (Figure 4d). Similarly, the shift of charge state distribution to higher charge state was also observed in angiotensinⅡ, which has a much smaller molecular weight (see Figure 4a vs. Figure 4b). Besides sodium ions, other cation species including K+, Mg2+ and Ca2+ were also investigated and similar phenomena of reduced nonspecific metal adduction to Leu-enkephalin were observed (see Figure S1). We also investigated the influence of analyte solvents, methanol/water and acetonitrile/water with different ratio was used to evaluate the influence on the metal ion adduction due to its widely use in ESI-MS. As was shown in Figure S2-S4, it was clear that no significant change in the sodiated ion distribution was observed when methanol/water with different ratio was used for ESI-MS, both for micro and nano emitter. Furthermore, the results obtained from nanoemitters also demonstrate that nanoemitters can efficiently decrease the abundance of the sodium adducted ions in all the cases where the amount of organic solvent ranged from 0% to 90%. Very similar phenomena were observed in acetonitrile (data not shown). Typically, the relative intensities of the sodium adducted ions were less than 5% (relative to the intensity of the protonated ions), see Figure S-2(d-f). These results clearly showed that the use of nanoemitters could be an efficient and universal approach to reduce the nonspecific metal adduction to peptide or protein ions in ESI-MS.

Mechanism of Reduced Metal Ion Adduction with nanoemitters As indicated by the preliminary experimental results and the previous reports, we suppose the mechanism of reduced metal ion adduction with nanoemitters is related to the physical properties of solution in the confined region inside the nanoemitters. Nanoemitter prepared from borosilicate glass capillaries here, could be viewed as a conical nanochannel with a nano-sized opening. The exclusion-enrichment effect (EEE) is one interesting aspect of the nanochannels. In aqueous solution, the silanol groups on the surface of the glass emitters can be deprotonated when the pH of the bulk solution is larger than 3, resulting in a negatively charged surface.31 An excess of counterions can be attracted to the near surface and co-ions can be excluded away due to electrostatic interactions with the surface charge, which was called exclusion enrichment effect, described by Plecis et al.22,23,32 In


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comparison with the bulk, an over two orders of magnitude times enrichment of oxonium ions in nanochannels (20 nm long and 20 nm wide) have been reported by numerical simulation.33 Kitamori et al. firstly demonstrated a super resolution laser induced fluorescence measurement technique to map the proton distribution in an extended nanochannel (400 nm wide) and the average proton concentration is 19.1 times higher than that of the bulk (pH = 6.22).34,35 This means the local pH inside the nanoemitter tip can be significantly lowered due to the EEE. A numerical simulation of the proton enrichment caused by the EEE in the emitter was also carried out by solving the multi-ion Poisson-Nernst-Planck (PNP) equation using a finite element analysis software package, COMSOL Multiphysics (version 5.2a), detail in SI. Figure S5 depicts that, in comparison with that of microemitter, a much higher degree of proton enrichment is obtained in nanoemitter. The proton concentration in microemitter dramatically decreases to be almost comparable with the bulk value (pH = 5.6) as the r/r0 varied from 1 (the glass surface) to 0 (the center of the emitter), see Figure S6. Due to the more significant the EDL overlapping effect, more protons are enriched in the nanoemitters, for example, the average proton concentration at the front end of the emitter was 0.09 mM for 120 nm nanoemitter, which was 35.8 times of bulk value (9.1 times of value in 1.2 µm microemitter). McLuckey et al. have reported that sodium adduction to protein ions could be significantly reduced when the solution pH is ∼3 units lower than the isoelectric point of the protein.36 The surface area relative to the solution volume in the nanoemitter tips increases as the tip size decreases. Therefore, better reducing effect can be expected as the tip size decreases, which have been confirmed by the experimental results summarized in Figure 2. Besides the EEE, the active transport of proton in nanochannels can also be contributive in the proton enrichment, because oxonium ions have a uniquely high mobility, a factor of ∼7 higher than sodium ions.37 It was found that, at low salt concentrations (∼5×10−6 M), the conduction of the nanochannels was dominated by oxonium ions.38 Kitamori et al. have also reported an enhancement of proton mobility phenomenon in extended-nanospace channels, and a three-phase model was used to elucidate this anomalous ion transport behavior in nanochannels.39 In the three-phase model, the solution near the surface was divided into three imaginary parts, that is, the bulk, absorbed, and intermediate phases. The so-called proton-transfer phase is an intermediate phase located in the nanochannel between the bulk phase and the absorbed phase, and it is believed to have played the key role in the enhanced proton-transfer mechanism. Protons could hop along a hydrogen-bond chain and the transport of charge defects could be promoted in the nanochannels by the rapid chemical exchange between H2O and SiOH groups on the glass surface.22,39,40 The existence of the proton-transfer phase was experimentally


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confirmed by NMR study41 and its thickness can be controlled by the salt concentrations.33,42-44 That is, the lower the salt concentrations are, the thicker the proton-transfer phase becomes. Thus, higher suppressing effect for sodium ion adduction was expected when the salt concentrations decrease, which was also supported by the experimental data summarized in Figure 2 and Figure 3. The average number of sodium ions in the adducts reduced from about 0.87±0.03 to 0.25±0.01 (reduced by 71 %) as the concentration of NaCl reduced from 10 mM to 1 mM (see Figure 3e vs. Figure 3d). Because of the thicker proton-transfer phase when decreased the salt concentration to 100 µM in nanoemitters, the average number of sodium ions in the adducts reduced dramatically to 0.03 (reduced by 88 %, Figure 2b vs. Figure 3d). To further estimate the influence of the strong external electric field (used here to generate electrospray) on proton enrichment, a simulation of the proton distribution was performed by coupling the EEE with the ion concentration polarization effect (ICP effect, caused by the presence of the external electric field). The results showed in Figure 5 revealed that a more significant enhancement of proton in the nanoemitter could be achieved by the presence of the external electric field. Taking the maximum proton concentration for interpretation, the maximum value of proton concentration is 0.38 mM (more than 150 folds of the bulk value) when the external electric field is applied, which is 2.8 times of the value where the external electric field is off. The ion concentration polarization along the height direction (z direction) yields, as expected, a remarkable influence on the enhancement of the proton concentration, especially in the tip region of the nanoemitter. As is shown in Figure 5c, the influence of the ICP effect is mainly focused on the first few hundreds of nanometers of the nanoemitter tip. It should be noted that the ICP effect is time-dependent, and the present simulation is a stationary one, thus the active transport of the proton is not included. But even in this simplified model, it is clear that the proton concentration inside a nanoemitter can be remarkably enhanced due to the EEE and ICP effect, yielding a lower local pH than that of a microemitter. To further evaluate that the solution acidification in the micro zone of the emitter tip can be attributable partially to the ICP effect, experiments were carried out using a preconcentration method with cytochrome c. A voltage of 1.0 kV was first applied with different time (no electrospraying could occur) and subsequent electrospray analysis was carried out by increasing the applied voltage to 3.5 kV. When a voltage of 1.0 kV was applied, solution polarization could occur and lead to the reduction of the local pH in the emitter tip due to the accumulation of oxonium ions. The conformation of cytochrome c can respond quickly to the change of surrounding pH and a shift of the charge state can be observed due to the conformation change (protein unfolding).21,31,45 As was shown in Figure S7, the charge state of


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cytochrome c increased remarkably from 6+ to 10+ (Figure S-7a) to 7+ to 18+ (Figure S-7f) as the preconcentration time increased from 0.1 min to 1.1 min. These results proved that the active transport of protons induced by the strong electrostatic field could also contribute to the reduction of pH value in the emitter tip. To further confirm that the reducing effect in metal ion adduction obtained with nanoemitters was a result of the acidification of solution in the micro zone of the emitter tip, the influence of solution pH was evaluated with Leu-enkephalin in acidic solutions using microemitters (~1.2 µm). As was shown in Figure S-9a, the pH was 3.6 and the most abundant molecular ions were regulated to be protonated ones, in good agreement with the previously reported results.36 However, it could not explain the disappearing of salt cluster peaks in Figure 3d, which was obtained with nanoemitters from a slightly acid solution (pH = 5.7, due to the absorption of ambient atmospheric carbon dioxide in de-ionized water). The disappearing of salt cluster peaks must be due to some other reasons. It was widely accepted that the size of the initial droplets should have a significant influence on the ion formation.3,46,47 Concentration enhancement could occur with the evaporation of solvents, and ions could release after a sequence of unsymmetrical droplet fissions. The origin of the different behavior between the microemitters and nanoemitters must lie in the process of solvent evaporation and droplet fission. The droplets generated from microemitters require more fission steps to give comparable charged droplet size of nanoemitters. It is usually assumed that with each offspring step the precursor droplet loses 15% of its charge and 2% of its mass.3,46 The uneven loss of charge and mass in the droplet fission leads to the formation of highly salt concentrated, but relatively lowly charged residue droplets, even the initial salt concentration is moderate (see Scheme 1). The observation of salt clusters in the high m/z region also presents strong evidence for the enrichment of salt during the process of solvent evaporation and droplet fission (see Figure S-8a). It was well known that droplets emitted from very small orifices are likely of reduced dimensions and highly charged,3,21 therefore, less fission steps is needed to release the high charged gas phrase ions (the charge state distribution shifted to higher charge state in nanoemitters, see Figure 4a vs. Figure 4b or Figure 4c vs. Figure 4d). That meant the enrichment effect of salt was not as significant as that of microemitters, which explained the disappearing of salt cluster peaks in the high m/z region (see Figure 3b and Figure S-8b). Herein, we preliminarily concluded the curious phenomenon of reduction of metal adduction formation in nanoemitters was a result of the proton-enriched smaller initial droplets.


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Comparison with the Method of Solution Additives and nESI Solution additives such as NH4Br12 and 18C615,49 have frequently been employed to promote the ionization of molecules of interests as well as eliminate the formation of metal adduct ions in ESI-MS. Adding 25 mM NH4Br into 10 µM Leu-enkephalin aqueous solution containing 1 mM NaCl have shown the ability in remarkably suppressing alkali ion adduction (Figure S-9b). The average number of sodium ions adducted to Leu-enkephalin reduced from 1.61±0.04 to 0.29±0.01 (see Figure 3a vs. Figure S-9b), but not as efficient as the use of nanoemitters (Figure 3d, the average number of sodium ions adducted to Leu-enkephalin was 0.25±0.01). The higher efficiency with nanoemitters in reducing the sodium ion adduction was further supported by the experimental fact that almost no di-sodiated ions [M+2Na-H]+ were observed in nanoemitters (see Figure 3d), but they are still discernible from salt cluster peaks in 25 mM NH4Br as solution additives (Figure S-9b). Besides, solution additives may suffer from the interference caused by the salt cluster peaks in the mass spectra, which was ineluctable in NH4Br as solution additives. It was obvious that a great abundance of salt clusters peaks were presented throughout the m/z region, peaks from molecular ions might be submerged. This could be even more severe if the resolution of the mass spectrometer was not sufficient to discriminate the molecular ions from the salt cluster ions. Crown ethers, such as 18C6, are another kind of solution additives, which can bind to alkali metal ions with high efficiency. Therefore, crown ethers have been utilized for reducing alkali ion adduction in ESI-MS.15,49 Figure S-9c showed the mass spectra acquired by adding 1 mM 18C6 into the aqueous solutions containing 10 µM Leu-enkephalin and 0.1 mM NaCl. Leu-enkephalin was detected mainly as 18C6 adduct [18C6+M+H]+ instead of sodiated ions. Although the sodium adduction was totally eliminated, the intensity of peaks from protonated ions was a much smaller fraction of the total ion intensity (0.25 %, relative to the total ion intensity of m/z range from 100 to 1000). Compared with methods using NH4Br and 18C6 as solution additives, the utilization of nanoemitters in ESI-MS may be a more efficient and easy-to-use approach to reduce metal ion adduction as well as enhance the matrix tolerance. Importantly, the charge balance inside the emitter could be realized via a bipolar spray process in PI-ESI. The anions left behind after the formation of the gas phase sodiated ions could be sprayed out during the off period of the pulsed ion source, see Figure S-10. The pulsed ion source should also have effects on the metal ion adduction. Therefore, we compared the results obtained using both PI-ESI and nESI to estimate the influence of the pulsed ion source. For microemitters, less sodium ion adducts trended to be formed in continuous ion source (nESI, Figure S-11a) than that of pulsed ion source


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(PI-ESI, Figure S-11c). A possible mechanism for the formation of massive sodium ion adducts in PI-ESI is the relatively lower amount of excess charge in the initial droplet when microemitters were used. Once electrospray occurred, the charge balance in nESI could be accomplished simultaneously by the electrochemical reactions happened on the interface between the electrode and solution, but this approach was not applicable for PI-ESI,48 because there was no physical contact between the solution and electrode in PI-ESI. Thus, the droplets formed in nESI could be more highly charged than that of PI-ESI. As a result, the droplet from PI-ESI could be more salt-enriched, because it needs more steps for desolvation to produce offspring droplets or ions, which are responsible for the production of sodiated ions. The signal intensity of analyte in PI-ESI was several times of lower than that of nESI, which also indicated that the initial droplet generated from PI-ESI was less charged than that of nESI. However, when nanoemitters were exploited, protonated ions were dominant both in continuous (nESI, Figure S-11b) and pulsed (PI-ESI, Figure S-11d) ion source, proving the efficiency of nanoemitters in suppressing the metal adduction in ESI-MS.

CONCLUSIONS In conclusion, a novel method has been developed to suppress the formation of metal adduct ions in ESI-MS by utilizing nanoemitters. The utilization of nanoemitters could significantly avoid the peak broadening caused by the multiple metal adducted ions, therefore boosted the analytical performance such as sensitivity. In comparison with that of microemitters, the strong solution acidification caused by the EEE and the active transport of the protons in nanoemitters was supposed to have played a pivotal role in the reduction of the nonspecific metal adduction to protein ions. The smaller droplets emitted from nanoemitters undergo less solvent evaporation and droplet fission steps. Thus, the enrichment of salt was not as significant as that of microemitters. This explained the disappearing of salt cluster peaks in the high m/z region. This method is characterized by ease of use and high matrix tolerance, no pre-treatment such as desalting process even in the presence of alkali salt at millimole concentration. Although the mechanism of the tuning of adduct ions is not fully understood, it does not hinder the application of this method, especially in cases where pre-treatment such as desalting can be really hard to carry out due to the ultra-small volume, e.g. single-cell samples. The present study demonstrates that nanoemitter holds much promise as a general means for eliminating the formation of unfavorable metal adduct ions in ESI-MS.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (J.J. Xu) Tel: +86-25-89687294 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation (21327902, 21535003) of China. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We also thank Prof. Wei Wang (Nanjing University) and Dr. Jinyi Li (Nanjing University) for insightful discussion and helpful suggestions in the simulation. ASSOCIATED CONTENT Supporting Information Additional experimental data and discussions as noted in text including the details regarding simulation are available free of charge on the ACS Publications website at: http://pubs.acs.org.


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Figure 1. Scanning electron micrographs of the emitters (vertical view) with different openings: (a) ~60 nm, (b) ~120 nm, (c) ~600 nm and (d) ~1200 nm.


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Figure 2. Mass spectra of 10 µM Leu-enkephalin aqueous solutions containing 100 µM NaCl obtained using PI-ESI from emitters with an opening of (a) ~60 nm, (b) ~120 nm (c) ~600 nm and (d) ~1200 nm.


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Figure 3. Mass spectra obtained using PI-ESI with microemitters (~1.2 µm) from 10 µM Leu-enkephalin aqueous solutions containing (a) 1 mM NaCl, (b) 10 mM NaCl, and (c) 25 mM NaCl. Mass spectra obtained using PI-ESI with nanoemitters (~120 nm) from 10 µM Leu-enkephalin aqueous solutions containing (d) 1 mM NaCl, (e) 10 mM NaCl and (f) 25 mM NaCl.


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Figure 4. Mass spectra of 10 µM angiotensin Ⅱ aqueous solutions containing 0.1 mM NaCl obtained from (a) ~1.2 µm microemitters and (c) ~120 nm nanoemitters; mass spectra of 10 ppm insulin (acetonitrile/water (50:50 vol.%)) solution containing 0.1 mM NaCl obtained with (b) ~1.2 µm microemitters, (d) ~120 nm nanoemitters.


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Figure 5. Proton distribution inside a 120 nm nanoemitter: (a) 2D axisymmetric illustration of the proton distribution, (b) proton distribution in the r direction (z = 0) at the front end of the nanoemitter, (c) proton distribution in the height direction (z direction, r = 0).


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Scheme 1. Schematic picture of the evolution of a charged droplet generated with microemitters and nanoemitters. The initial droplets from nanoemitters are smaller and proton enriched due to the EEE and the active transport of protons. The offspring droplets from microemitters (comparable with the initial droplets from nanoemitters in size) are salt concentrated and more likely to generate sodiated protein ions and salt cluster ions.


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Effect of Nanoemitters on Suppressing the ... - ACS Publications

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