Peptide Fragmentation during Nanoelectrospray Ionization - Analytical

Anal. Chem. 2010, 82, 6534–6541

Peptide Fragmentation during Nanoelectrospray Ionization He Wang,† Zheng Ouyang,† and Yu Xia*,‡ Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907-2032, and Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393 Electrospray ionization (ESI) is considered a soft ionization method, and typically no peptide ion fragmentation is observed. Recently, it has been observed that intensive fragmentation of peptide ions can occur during nanoelectrospray (nanoESI) at special conditions, such as solutions containing high concentrations of salt and relatively low voltage for the spray. In this study, peptide fragmentation during nanoESI has been systematically characterized. The fragmentation phenomenon was observed for a variety of peptides with molecular weights lower than 3000 Da and the types of fragments include a, b, and y ions. For phosphorylated peptides, very little loss of the labile phosphate groups was observed. Solution electrical conductivity (K) and flow rate were identified as the key parameters affecting the degree of peptide fragmentation. Mechanistic studies suggested that very fine first generation charged droplets (∼30 nm in diameter) with high surface electric field (∼1 V/nm) could be formed from nanoESI of highly conductive solutions (K ) 0.4 S/m) and at a low flow rate (2 nL/min). It is proposed that solvated peptide ions are ejected with high kinetic energies from the early generations of charged droplets, and the subsequent collisional activation in air induces the peptide fragmentation. The relatively high degree of solvation around the phosphate groups may contribute to the preservation of the phosphorylation during the activation process. The capability of forming intact ions of nonvolatile molecules by electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization (MALDI)2 has significantly enlarged the scope of mass spectrometry (MS) in biological studies. Taking ESI-MS as an example, the molecular weight information of a biomolecule can be obtained from the multiple charge states of the protonated or deprotonated forms of the molecule, depending on the ionization mode of ESI.3 Structural information of a biomolecule, e.g., a peptide, can be acquired by combining ESI with a number of activation * To whom correspondence should be addressed. Phone: (765) 494-1142. Fax: (765) 494-0239. E-mail: [email protected]. † Weldon School of Biomedical Engineering. ‡ Department of Chemistry. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37–70.

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methods within the context of tandem mass spectrometry,4,5 which is used as an essential analytical tool in proteomics.6,7 While ESI-MS is routinely applied for biochemical analysis, the interest in understanding the fundamental aspects of ESI remains strong, not only because the ESI process plays a crucial role in terms of relating the properties of the gas-phase ions to the original molecules in solutions but also because fundamentally it is a complicated and intriguing chemical process. The process of ESI is generally accepted as following: a high voltage applied in ESI forms a strong electric field at the tip of the emitter, which forces the electrified liquid to extend and form a cone (Taylor cone); the tip of the cone is further extended to a liquid filament under the electric force; eventually, unbalance between the electric field and the solution surface tension causes the breakup of the filament into charged droplets.8 The charged residue model (CRM)9 and the ion evaporation model (IEM) are widely accepted to account for the formation of free gas-phase ions from the charged droplets.10,11 The charged residue model is based on the assumption that after a series of solvent evaporation and Coulomb explosions, the fine charged droplets each containing only one solute molecule are produced. Free gas-phase ions are generated after the evaporation of the rest solvent molecules. This mechanism has been tested experimentally and is applied for the formation of multiply charged ions from molecules with molecular weight above 3000 Da.12,13 The ion evaporation model, proposed by Iribarine and Thomson, argues that solvated ions can be ejected from the charged droplets when the droplet size is relatively small ( 0.4 S/m). Note that this K value is 4 orders of magnitude higher than that with deionized water as the solvent and 40 times higher than that with MeOH/H2O/HOAc (50/49/1, v/v/v) (K ) 0.01 S/m). Effect of Solution pH. The data in Figure 2B show that sulfuric acid produces the highest frag% for nanoESI of aqueous peptide solutions of the same conductivity. Given the strong acidity of sulfuric acid, the effect of the solution pH on the degree of peptide fragmentation during nanoESI needs to be considered. The pH values of solutions containing 200 mM NH4HCO2 were adjusted from pH ) 2 to pH ) 11 by adding appropriate amounts of NH4OH. Bradykinin 2-9 (100 µM) was dissolved in these solutions that were sprayed using bulk-loaded nanoESI. The frag% was plotted as a function of the solution pH values (Figure S-4 in the Supporting Information). Surprisingly a relatively constant frag% (∼27%) was obtained across the whole range of the pH value from 2 to 11. This set of experiments suggest that the peptide fragmentation during nanoESI is not affected by the solution

Table 1. List of the Frag% of Bradykinin 2-9 (100 µM) Using Different Solvent Systems Containing 50 mM H2SO4 for Bulk-Loaded nanoESI

Figure 3. Plots of the frag% of bradykinin 2-9 (PPGFSPFR, 100 µM) in aqueous solution containing 100 mM NH4OAc as a function of solution flow rates with different sizes of the spray tip opening: black curve, 5 ( 1 µm.; red curve, 8 ( 1 µm; blue curve, 15 ( 1 µm; green curve, 30 ( 2 µm. Error bars represent standard deviations acquired by evaluating three spectra collected at the same conditions.

pH values while the high fragmentation percentage associated with sulfuric acid requires further investigation. Effect of Spray Flow Rate and the Size of Spray Tip Opening. In the bulk-loaded nanoESI setup, the solution flow is driven by electric field and capillary force. Under the conditions optimized for peptide fragmentation, the flow rate (Vf) was determined to be 2 nL/min based on the volume consumption measured within a given time (average of three measurements). In order to systematically vary the solution flow rate and the size of tip opening, a continuous-flow nanoESI setup was employed instead of the bulk-loaded source. An uncoated fused silica capillary with tapered spray tip (New Objective, Woburn, MA) was connected to a syringe pump that was used to control the solution flow. A series of sizes (inner diameter, i.d.) of the tip opening was tested, including 5 ± 1, 8 ± 1, 15 ± 1, and 30 ± 2 µm. An aqueous solution containing 100 µM bradykinin 2-9 and 100 mM NH4OAc was used for this set of experiments. The frag% of the peptide was plotted as a function of the solution flow rates for different sizes of the tip openings, as shown in Figure 3. When the tip size is relatively large, i.e., 30 µm, no fragmentation can be observed across the tested range of the flow rates (100-600 nL/min, no stable spray signal for a flow rate lower than 100 nL/min). For smaller tip openings (8 and 15 µm), stable fragmentation can be observed during nanoESI, however only when the flow rates are below certain thresholds. When the tip opening is further decreased to 5 µm, stable and intense fragmentation of the peptide can be observed for the range of the flow rates tested. These results show that the degree of peptide fragmentation is affected collectively by the size of the spray tip opening and the solution flow rate. Furthermore, peptide fragmentation is enhanced with decreases in both tip sizes and flow rates. These findings are consistent with that observed using the bulk-loaded nanoESI setup. In the latter case, the sizes of the tip opening are typically smaller than 20 µm and the flow rate is about 2 nL/min, both of which fall within the range favorable for observing peptide fragmentation. It was also consistently observed with the continuous nanoESI source that no stable fragmentation could occur when the electrical conductivities of peptide solutions were relatively low. Effect of Solvent Surface Tension and Polarity. Other than using water, solvents with different physical properties, e.g., polarity and surface tension, were investigated. To maintain a

solvent

frag%

γ (25 °C, mN/m)

ε

methanol acetone tetrahydrofuran chloroform acetonitrile water

61% 51% 42% 51% 48% 57%

22.07 22.72 26.40 26.67 28.66 71.99

33 20.7 7.5 4.8 37 80

relatively high solution electrical conductivity, 50 mM H2SO4 was added to each solution containing 100 µM bradykinin 2-9. The solution surface tension (γ) and dielectric constant (ε) for the pure solvents are listed in Table 1 together with the observed frag% of bradykinin 2-9 under each solvent conditions. The corresponding nanoESI spectra are provided in Figure S-5 in the Supporting Information. For the range of solution surface tension (22-72 mN/m) tested herein, no clear trend can be observed with regard to the frag% of the peptide. Dielectric constant can be used as one of the parameters to evaluate the degree of polarity of different solvents. Water is the most polar and chloroform is the least polar solvent in our studies. Quite extensive peptide fragmentation was observed in both conditions. No correlation between the peptide frag% and the dielectric constant can be drawn from the data summarized in Table 1. Effect of Spray Voltage. The spray voltage was found to be critical in observing peptide fragmentation during nanoESI. Figure 4A shows the plot of the frag% of badykinin 2-9 (100 µM in aqueous solution containing 100 mM NH4OAc) as a function of the spray voltage. The spray voltage was increased from 900 to 1300 V with a step of increment of 50 V. The spray currents were measured correspondingly and plotted in Figure 4A. The spray started when the voltage was increased to 950 V, and peptide fragmentation was observed simultaneously. The frag% of the peptide was relatively stable when the spray voltage was below 1100 V. In this range of the spray voltage applied, the spray currents were about 1 nA. A typical nanoESI spectrum is shown

Figure 4. (A) Plots of the frag% of bradykinin 2-9 (100 µM in aqueous solution containing 100 mM NH4OAc) (red curve) and the spray current (black curve) as functions of the applied voltages using bulk-loaded nanoESI. MS spectra of the same solution when the spray voltage was set at (B) 1.0 kV (spray current, 1 nA) and (C) 1.25 kV (spray current, 90 nA). Analytical Chemistry, Vol. 82, No. 15, August 1, 2010

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in Figure 4B with the spray voltage set at 1.0 kV. As the spray voltage was further increased, the spray current increased monotonically while the frag% decreased. When the spray voltage was above 1200 V, almost no fragmentation could be observed (Figure 4C). The spray current (as a function of time) at each spray voltage was stable and there was no observed proof of the change of the spray mode from a pulsed spray to cone jet spray, which was described by Vertes group in their studies.26 The distance between the tip and the inlet of the mass spectrometer did not show an obvious effect on the fragmentation of the peptide when an optimized spray voltage was applied. Mechanism. The nanoESI source is an electrolytic cell, where electrolysis or hydrolysis can occur and induce peptide backbone cleavages in the solution phase.27 In an experiment to verify this, an aqueous solution containing 10 µM bradykinin 2-9 and 100 mM NH4OAc was subjected to 100 µA electric current for 30 s and then analyzed by both MALDI-TOF-MS and ESI-MS. No peptide fragments were observed, which suggested that electrolysis was not responsible for the peptide fragmentation observed during nanoESI. The interface condition of a mass spectrometer can be tuned to provide ion activation to cause ion desolvation, typically but also ion fragmentation with an interface condition harsh enough.21,28 For all the experiments performed in this study, the interface condition of the LTQ mass spectrometer was adjusted so that little peptide fragmentation was observed during nanoESI of peptide samples using low conductivity solutions. Another possible source of fragmentation at atmospheric pressure would be an electrical discharge caused by a strong local electric field at the spray tip or the emission of highly energetic small ions directly from the Taylor cone. Since peptide fragmentation is typically observed with low spray voltage (∼1 kV) and small spray current (∼1 nA), while no peptide ion fragmentation is observed under higher voltages (Figure 4), the likelihood for fragmentation caused by discharge is small. In addition, the similar peptide fragmentation observed from using organic solvent with low surface tension as compared to that from aqueous solution (Table 1) does not support that discharge is induced under these conditions. In order to verify if peptide fragmentation occurred during the ionization process before the analytes reached the mass spectrometer interface, the sprayed plume was collected and analyzed. A gold plate (grounded) was used as the counter electrode for nanoESI of bradykinin 2-9 (100 µM in aqueous solution containing 100 mM NH4OAc, spray voltage 1 kV, optimized for fragmentation). After 10 h of collection, the condensate spot was washed with 10 µL of methanol and followed by MALDI-TOF analysis. The MALDI-TOF condition was adjusted using a standard bradykinin 2-9 sample to minimize any in-source fragmentation, as shown in Figure 5A. The MALDI-TOF spectrum of the spray condensate from nanoESI of high conductivity solution is shown in Figure 5B. Peptide backbone fragment ions can be clearly seen, such as y5-y7 ions and their oxidation products (marked with “*”). The oxidation peaks have mass increases of 14 and 16 Da, which are possibly due to oxidation at the amino acid (26) Nemes, P.; Marginean, I.; Vertes, A. Anal. Chem. 2007, 79, 3105–3116. (27) Jackson, G. S.; Enke, C. G. Anal. Chem. 1999, 71, 3777–3784. (28) Loo, J. A.; Udseth, H. R.; Smith, R. D.; Futrell, J. H. Rapid Commun. Mass Spectrom. 1988, 2, 207–210.

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Figure 5. Positive ion mode MALDI-TOF MS spectra of (A) bradykinin 2-9 and (B) the condensate collected from bulk-loaded nanoESI of 100 µM of bradykinin 2-9 in aqueous solution containing 100 mM NH4OAc.

residue side chains after long time exposure in air during the collection process.29 Nonetheless, this set of experiments supports the hypothesis that peptide fragmentation happens during the ionization process before them entering the mass spectrometer. Several groups have shown that an increased solution conductivity leads to a smaller radius of the charged droplet,16,30 and the following equations have been established for estimating the radius (R) and the maximum electric field (E) of the first generation charged droplets:14,16

( )

(1)

()

(2)

R ) G(ε)

E)

Vfεε0 K

γ1/2 K ε02/3 Vf

1/3

1/6

where the G(ε) is a coefficient related to the dielectric constant of the solution, ε is solution dielectric constant, ε0 is the vacuum permittivity, K is solution electrical conductivity, and Vf stands for the flow rate. For the experimental conditions with which peptide ion fragmentation is typically observed with water as the solvent, e.g., Vf ) 2 nL/min, K ) 0.4 S/m, and G(ε) ) 0.85,31 the size of the first generation charged droplets is calculated around 30 nm and the maximum electric field is 0.95 V/nm, based on eqs 1 and 2. Note that the scaling laws were established for ESI of polar liquids in the cone-jet mode, where the currents measured in those systems were 1 or 2 orders of magnitude higher than that measured in the current study.31,32 The application of the scaling laws to the current system may need to be further evaluated. The charges on the surface of the droplet can be readily calculated using Coulomb’s law (eq 3): q ) E4πε0R2 (29) (30) (31) (32)

(3)

Xu, G. H.; Chance, M. R. Chem. Rev. 2007, 107, 3514–3543. Kebarle, P.; Peschke, M. Anal. Chim. Acta 2000, 406, 11–35. Chen, D. R.; Pui, D. Y. H. Aerosol Sci. Technol. 1997, 27, 367–380. de la Mora, J. F.; Locertales, I. G. J. Fluid Mech. 1994, 260, 155–184.

The amount of charges is about 90% of the charges needed for the same size charged droplet to reach the Rayleigh limit, which can be calculated using eq 4:33 qRy ) 8π(ε0γR3)1/2

(4)

For the peptides tested in this study, of which the molecular weights are all lower than 3000 Da, the ionization process presumably should be characterized by the ion evaporation model. Although no literature data are available to establish the lowest electric field for the evaporation of solvated peptide ions, experiments and calculations on small organic/inorganic ions show that ion evaporation can start when the electric field is in the range of 1 V/nm.11,14,15 With a surface electric field of 0.95 V/nm and a charge level of 90% of the Rayleigh limit for the first generation charged droplets, ion evaporation of the solvated peptide ions can be competitive with Coulomb fission. The microsolvated ions on the surface of the charged droplet should be ejected with a kinetic energy defined by the droplet electric field. The mean free path in air under the standard condition is about 66 nm.34 Although the electric field decreases quickly away from the charged droplet surface, a doubly charged microsolvated peptide ion may still gain tens of electrovolts before the first collision event. The amount of kinetic energy might be high enough to induce cleavages of the peptide backbone under multiple collision conditions. This hypothesis is supported by the fact that the peptide fragments can be collected outside the mass spectrometer (Figure 5B). In addition, very similar peptide fragmentation as that in nanoESI was observed in desorption electrospray ionization (DESI)35 of peptides using a high conductivity solution as the spray solvent (Figure S-6 in the Supporting Information). Since in DESI the desorption and ionization processes are separated, peptide ions are formed from secondary charged droplets instead of the Taylor cone. This result indicated that ion evaporation from charged droplets should be one of the mechanisms accounting for peptide fragmentation observed in nanoESI. It has been demonstrated previously that direct ion emission from the Taylor cone can be achieved by spraying highly conductive solutions, e.g., ionic liquid solutions (K ) 1-3 S/m), at low flow rates in vacuum.36,37 However, because of the dramatic difference in the analyte/solvent system used in this study, it remains unclear if direct ion emission from the bulk solution would apply to the findings described herein. One could argue that the high kinetic energy gain occurs in all peptide ions formed from the ion evaporation model, while no fragments are seen under typical nanoESI conditions. For a comparison, the size, surface electric field, and the charges on the first generation droplets formed from nanoESI of a low conductivity solvent, i.e., deionized water (K ∼ 4.0 × 10-5 S/m), are calculated and listed as condition 2 in Table 2. The radius of the first generation charged droplet is calculated to be 645 nm, (33) Rayleigh, L. Philos. Mag. 1882, 14, 184–186. (34) Serway, R. Physics for Scientists and Engineers with Modern Physics, 3rd ed; Saunders College Publishing: Philadelphia, PA, 1990. (35) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (36) Romero-Sanz, I.; Bocanegra, R.; de la Mora, J. F.; Gamero-Castano, M. J. Appl. Phys. 2003, 94, 3599–3605. (37) Garoz, D.; Bueno, C.; Larriba, C.; Castro, S.; Romero-Sanz, I.; de la Mora, J. F.; Yoshida, Y.; Saito, G. J. Appl. Phys. 2007, 102, 064913.

Table 2. Characteristics of the First Generation Charged Droplets Formed from NanoESI of Aqueous Solutions under Different Conditionsa parameters

condition 1

condition 2

condition 3

electrical conductivity (K, S/m) flow rate (Vf, nL/min) initial droplet size (R, nm) electric field (E, V/nm) no. of unit charges Rayleigh charge limit

0.4 2 32 0.95 593 655

4.0 × 10-5 2 645 0.20 57 400 67 600

0.4 20 70 0.65 2146 2285

a Condition 1: aqueous solution containing 100 mM NH4OAc, spray voltage optimized for peptide fragmentation (1.1 kV). Condition 2: pure water solution, no peptide fragmentation observed. Condition 3: aqueous solution containing 100 mM NH4OAc, spray voltage set at 1.5 kV, no peptide fragmentation observed.

which is 20 times larger than that formed from a solution with K at 0.4 S/m. Since the number of charges (57 400 unit charges) and the electric field (0.20 V/nm) are much smaller than those required for Coulomb explosion or ion evaporation, the first generation charged droplets undergo solvent evaporation until reaching the Rayleigh limit. If 2% masses and 15% charges of the initial charged droplet are distributed to 20 droplets upon Coulomb explosion,8 these fine droplets will each have 431 elementary charges, a radius of 58 nm, and a surface electric field of 0.18 V/nm. These droplets have to shrink to a radius of 25 nm to reach a surface electric field of 1 V/nm, which might be enough for ion evaporation. On the basis of the above estimations, a significant amount of solvent evaporation is required before the formation of gas-phase peptide ions from the first generation charged droplets of submicrometer sizes. The evolution process for the charged droplets is schematically presented in Figure S-7 in the Supporting Information. Note that for nanoESI of high conductivity solutions, ions are proposed to be ejected from the early generations of charged droplets without solvent evaporation. Solvent evaporation has been shown to affect the internal energies of the ions formed in ESI. Collette et al. demonstrated that a higher vapor pressure solvent system produced ions with an average internal energy 0.5 eV lower than that from a lower vapor pressure system in ESI.38 It is possible that in our systems solvent evaporation lowers the internal energies of the peptide ions formed from large initial charged droplets as compared to ions formed from charged droplets without solvent evaporation. Therefore, the similar collisional activation might not provide enough energy to vibrationally excite the ions to the level for backbone fragmentation (activation energy for amide bond cleavages, ∼1.2 eV).39-41 It is worth pointing out that although collisional activation might be responsible for the peptide fragmentation, the fragmentation patterns observed from nanoESI of high conductivity solutions are very different from those typically observed from ion-trap CID of the isolated peptide ions. One good example is that very little loss of phosphorylation is observed when phosphopeptides are dissociated during nanoESI (Figures 1B and (38) Collette, C.; De Pauw, E. Rapid Commun. Mass Spectrom. 1998, 12, 165– 170. (39) Busman, M.; Rockwood, A. L.; Smith, R. D. J. Phys. Chem. 1992, 96, 2397– 2400. (40) Meotner, M.; Dongre, A. R.; Somogyi, A.; Wysocki, V. H. Rapid Commun. Mass Spectrom. 1995, 9, 829–836. (41) Butcher, D. J.; Asano, K. G.; Goeringer, D. E.; McLuckey, S. A. J. Phys. Chem. A 1999, 103, 8664–8671.

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Figure S-1C in the Supporting Information), while the loss of phosphate groups is dominant during the ion-trap CID of the protonated peptide ions (inset of Figure 1). The differences in the fragmentation patterns are contributed by the differences in energy deposition and the types of ionic species that undergoes activation. During the ion-trap CID process, the dry peptide ions are subjected to multiple low-energy collisions. Because of the slow heating nature,42 loss of phosphorylation is the dominant process owing to its low activation energy (∼0.5 eV)43,44 as compared to peptide amide bond cleavage (∼1.2 eV).39-41 For peptide fragmentation during nanoESI, the collision energy (∼100 eV) is much higher than that of ion-trap CID (less than 20 eV for each collision event) and only the first several collisions are likely to contribute to the activation process due to the fast collisional cooling rates at atmospheric pressure. Furthermore, it is the microsolvated ions that are subjected to the collisional activation, which is different from the dry peptide ions for the ion-trap CID. The relatively heavy solvation around the polar groups, e.g., phosphate groups, might also result in a protection of the labile phosphorylation while rich peptide backbone fragmentation is induced. We also found similarities in peptide fragmentation from nanoESI and that induced in the skimmer-tube lens region (insource CID) in terms of fragment identities and their relative abundances (compare parts C and D of Figure S-2 in the Supporting Information). For phosphopeptides, a reduced loss of phosphate groups was generally observed for in-source CID as compared to ion-trap CID, although the highest degree of phosphate group preservation was found from peptide fragmentation in nanoESI (compare Figure S-8 in the Supporting Information to Figure 1). In addition to dried peptide ions, solvated ions and charged droplets are the major species subjected to beam-type collisional activation during in-source CID. The similarities of peptide fragmentation patterns observed in nanoESI and in-source CID may well suggest that similar population of species undergo similar ion activation under these two conditions. Note that studies on the MS/MS of solvated peptide ions are needed to further elucidate the proposed mechanism. The importance of producing small charged droplets at the initial stage of spray ionization to observing peptide ion fragmentation is supported by the experimental data. As shown in Figure 2B, for a given size of the nanoESI tip opening, e.g., 15 µm, the frag% of the peptide is largely reduced at higher solution flow rates (Vf). According to eqs 1 and 2, the increased flow rate leads to the larger sizes and smaller surface electric field of the charged droplets generated. Under those conditions, solvent evaporation occurs before ion evaporation and thus quenches the fragmentation. This scenario also explains that no fragmentation is observed when the nanoESI tip opening is too big (30 µm), where larger initial charged droplets are formed, presumably. Equation 2 predicts that higher solution surface tension leads to stronger electric field on the solution surface, which is expected to enhance the ion evaporation. However, for the range of the surface tension tested in this study (23-72 mN/m), no significant effect has been observed for the frag% of the peptide ions (as shown in Table 1). (42) McLuckey, S. A.; Goeringer, D. E. J. Mass. Spectrom. 1997, 32, 461–474. (43) Flora, J. W.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2004, 15, 121– 127. (44) Gronert, S.; Li, K. H.; Horiuchi, M. J. Am. Soc. Mass Spectrom. 2005, 16, 1905–1914.

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It is also interesting that the degree of peptide fragmentation is affected by the spray potential, as demonstrated in Figure 4. Note that the fragmentation is only observed under low spray voltage (∼1000 V) and small spray current (1 nA). When the voltage is increased above a certain threshold, i.e., 1200 V, no fragmentation is observed and there is a steep increase of the spray current. de la Mora and Locertales have shown that the spray current increases with the square root of the flow rate.32 Therefore, the large spray currents observed at relatively high spray voltages should correspond to the increased solution flow rates. In fact, a flow rate of 20 nL/min was measured for spray at 1.5 kV, which is 10 times higher than that for 1 kV, and the peptide fragmentation was no longer observed. Under this flow rate, a larger size (∼70 nm) of the initial charged droplets is formed with relatively low surface electric field (0.65 V/nm) (condition 3 in Table 2). The size of the peptide could also be a factor which affects the degree of fragmentation during nanoESI. For peptides with molecular weight lower than 3 kDa tested in this study, they all gave detectable fragmentation. We have not studied peptides with molecular weight within the range of 3-8 kDa. However, for two small proteins, i.e., ubiquitin (8.5 kDa) and cytochrome c (12 kDa), no fragmentation was observed under the nanoESI conditions used for peptide fragmentation. The discrepancy may come from that spray ionization of proteins undergoes different process as suggested by the charge residue model, which does not involve ion ejection under a high electric field. If this was the case, the ion evaporation model and the charge residue model could be potentially differentiated by examining the degree of ion fragmentation under nanoESI of highly conductive solutions. CONCLUSIONS In this study, we demonstrated that intense peptide fragmentation could be induced during nanoESI of high conductivity solutions. The degree of peptide fragmentation is promoted by high solution electrical conductivity and low flow rate, which are critical in producing fine, initial charged droplets. NanoESI of a conductive aqueous solution (K ) 0.4 S/m) at a flow rate of 2 nL/min are estimated to form the first initial generation droplets with small sizes (∼30 nm) and high surface electric fields (∼1 V/nm). Microsolvated peptide ions are proposed to be ejected from those fine charged droplets with high kinetic energies. The following collisional activation is responsible for inducing the peptide fragmentation. However, it is important to distinguish the above process from peptide ions derived from large-size initial charged droplets, e.g., ESI of low conductivity solutions. In the latter situation, a significant amount of solvent evaporation is needed before ion evaporation occurs, which may reduce the internal energies of the charged droplets and ions and subsequently limit the peptide fragmentation. It is worth pointing out that the fragmentation patterns obtained from nanoESI of high conductivity solutions are very different from those for ion-trap CID. It is suggested that the difference comes from the fast collisional activation of the microsolvated ions in ambient air as compared to the slow heating of dry ions inside an ion trap. The higher degree of solvation around polar groups, i.e., phosphate groups, may serve a role of protecting the labile phosphorylation during activation. This feature is especially attractive with respect to phosphopeptide characterization. Our study supports the ion

evaporation model for the ionization of small peptides using ESI and that solvent evaporation strongly affects the “softness” of ESI.38

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT H.W. and Z.O. acknowledge support by the National Science Foundation (Grant CHE 0847205).

Received for review April 2, 2010. Accepted June 25, 2010. AC100872X

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