B American Society for Mass Spectrometry, 2017 J. Am. Soc. Mass Spectrom. (2017) 28:2160Y2169 DOI: /s RESEARCH ARTICLE

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1 B American Society for Mass Spectrometry, 2017 J. Am. Soc. Mass Spectrom. (2017) 28:2160Y2169 DOI: /s RESEARCH ARTICLE Variables Affecting the Internal Energy of Peptide Ions During Separation by Differential Ion Mobility Spectrometry Brandon G. Santiago, Matthew T. Campbell, Gary L. Glish Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Campus Box 3290, Chapel Hill, NC , USA Abstract. Differential ion mobility spectrometry (DIMS) devices separate ions on the basis of differences in ion mobility in low and high electric fields, and can be used as a stand-alone analytical method or as a separation step before further analysis. As with other ion mobility separation techniques, the ability of DIMS separations to retain the structural characteristics of analytes has been of concern. For DIMS separations, this potential loss of ion structure originates from the fact that the separations occur at atmospheric pressure and the ions, during their transit through the device, undergo repeated collisions with the DIMS carrier gas while being accelerated by the electric field. These collisions have the ability to increase the internal energy distribution of the ions, which can cause isomerization or fragmentation. The increase in internal energy of the ions is based on a number of variables, including the dispersion field and characteristics of the carrier gas such as temperature and composition. The effects of these parameters on the intra-dims fragmentation of multiply charged ions of the peptides bradykinin (RPPGFSPFR) and GLISH are discussed herein. Furthermore, similarities and differences in the internal energy deposition that occur during collisional activation in tandem mass spectrometry experiments are discussed, as the fragmentation pathways accessed by both are similar. Keywords: Ion mobility, Fragmentation, Internal energy, Peptide Received: 24 January 2017/Revised: 7 May 2017/Accepted: 22 May 2017/Published Online: 26 June 2017 Introduction Ion mobility separations are a group of gas-phase techniques that can be implemented after ionization to separate ions on the basis of structural differences; however, the ability of these separation techniques to fully retain the structural attributes of ions has been the subject of much investigation. Most of the studies examining the preservation of structure have attempted to either minimize the amount of internal energy deposited into ions or to study the change in effective temperature of the ion [1 5]. Although most ion mobility techniques involve separation of ions in time with a low electric field, usually tens to hundreds of volts per centimeter, differential ion mobility spectrometry (DIMS) separates ions in space on the basis of Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. Correspondence to: Gary Glish; glish@unc.edu differences in their mobilities in alternating low and high electric fields (more than 10 kv/cm). A DIMS device consists of two parallel electrodes separated by a gap through which ions and a carrier gas flow. An asymmetric waveform is applied across the gap, alternating between high and low electric field strengths of opposite polarities on microsecond timescales. The amplitude of this waveform (V 0 P )isdefinedas the dispersion field (E D ), and a common method to improve DIMS separations is to increase the E D applied [2, 6 8]. After the ions enter the DIMS device, the ion movement toward the exit of the DIMS device is controlled by the carrier gas flow; however, the applied electric field accelerates ions orthogonal to the carrier gas flow, toward one or the other electrodes, on the basis of the polarity of the dispersion waveform applied. The ion motion toward an electrode during the low-field portion of the waveform is determined by the product of the electric field strength (E L ), the low-field mobility of the ion (K L ), and the time spent in the low field (t L ). When the waveform switches polarity and amplitude, the motion toward the opposite electrode during the high-field portion of the waveform is determined by the product of the electric field strength (E H ), the high-

2 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition 2161 field mobility of the ion (K H ), and the time spent in the high field (t H ). Use of a waveform with E L t L = E H t H yields the ion s net displacement for each period of the waveform to be proportional to the difference between its high-field and low-field mobilities (K H K L ). This net displacement is integrated across the ion s transit through the DIMS device, and ions are separated in space by the differences in their net displacement toward one of the electrodes. The net displacement during each period of the waveform can be made zero through the application of a DC compensation field (E C ). At a given E C, ions of only a selected differential mobility pass through the device, whereas ions with other differential mobilities are neutralized on the DIMS electrodes. Percent transmission of the selected ion through a DIMS device is a complicated function of many parameters, including K H, K L, diffusion coefficients, and the device dimensions, and can significantly decrease ion signal. To address this issue we designed a flare into the exit of our DIMS devices, and this can substantially improve transmission, with transmissions as high as 50% [9, 10]. The ion motion occurs at atmospheric pressure in DIMS devices, and therefore the ions undergo repeated collisions with the DIMS carrier gas [6, 11, 12]. The acceleration of the ions due to the dispersion field causes these collisions with the carrier gas to increase the internal energy of the ion. The ion velocity and the gas number density, along with the carrier gas temperature and identity, affect the ion molecule interactions that occur within the DIMS device and ultimately determine the amount of internal energy deposited into the ion during transit through the device [13 15]. The extent of the collisional heating has been studied by various approaches, including attempts to determine the effective temperature of the ions experimentally and comparisons with heating in drift-tube ion mobility spectrometers [2, 3, 16, 17]. Previously reported work has also shown that collisions in a DIMS device can impart enough internal energy to cause isomerization of multiply charged protein ions and fragmentation of ions formed from small molecules and proton-bound dimers [2, 3, 13 16, 18, 19]. However, there have been no reports of intra-dims fragmentation of larger ions such as peptides. The fragmentation of these larger ions would be less likely because of the greater number of degrees of freedom in larger ions over which the internal energy can be distributed. A schematic representation of this intra-dims fragmentation is shown in Figure 1, where a triply charged ion enters the DIMS device while an E C of 150 V/cm is applied to the DIMS device. In Figure 1a, thee C does not sufficiently compensate for the trajectory of the triply charged ion, and after some number of dispersion field waveform cycles, the ion collides with an electrode and is neutralized. This is representative of an ion without sufficient internal energy to undergo fragmentation in the DIMS device. An E C of 150 V/cm does correct for the trajectory of the doubly charged ion shown in Figure 1b,which is formed before it enters the DIMS device. In Figure 1c the internal energy of the triply charged ion is increased enough in the DIMS device that fragmentation can occur before the ion is neutralized. In Figure 1c the triply charged ion fragments into a doubly charged ion and a singly charged ion. The newly (a) (b) (c) X 1+ To MS formed species will then travel through the DIMS device on the basis of their own differential ion mobility and will be detected at the characteristic E C that permits that species to stably move through the DIMS device and into the mass spectrometer. This characteristic E C is the same E C that passes the ion through the DIMS device and into the mass spectrometer when the ion is formed before it enters the DIMS device. This is shown in Figure 1b and c, where a compensation field of 150 V/cm corrects for the trajectory of the doubly charged ion formed before it enters the DIMS device and the doubly charged ion formed in the DIMS device. An alternative example might have the charge retained entirely by one fragment ion, with a neutral fragment traveling through the DIMS device carried by the gas flow and unaffected by the dispersion field. The signal detected for each of the fragment ions is not solely based on the amount of fragment formed. Other factors, including the diffusion of the fragment ion, the mobility of the fragment ion in low and high fields, and the difference between the characteristic E C of the parent ion and the fragment ion, affect the signal detected. In this work the intra-dims fragmentation of multiply charged peptides along with the similarities and differences X EC= 150 V/cm EC= 150 V/cm EC= 150 V/cm To MS To MS Figure 1. a A compensation field of 150 V/cm does not correct for the trajectory of the triply charged ion and it is neutralized. b A compensation field of 150 V/cm does correct for the trajectory of the doubly charged ion. c The triply charge ion undergoes intra-differential ion mobility spectrometry (DIMS) fragmentation, and the doubly charged fragment ion stably passes through the DIMS device. MS mass spectrometer

3 2162 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition between intra-dims activation and the collisional activation step of collision-induced dissociation (CID) are discussed. The increase in internal energy is shown to occur via a slow heating process analogous to the collisional activation step despite the voltages and pressures used being vastly different [16, 20]. Those similarities are used to determine from which parent ion fragments originate. The effects on intra-dims fragmentation of changing the temperature of the DIMS carrier gas are also described. Additionally, studies in which the amount of internal energy added to the ions changes as a function of the DIMS carrier gas composition are reported. The influence of both temperature and carrier gas composition is also discussed in regard to the influence on the separation power of the DIMS analysis. Experimental Methanol (Optima grade), water (high-performance liquid chromatography grade), and acetic acid (ACS plus) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Bradykinin (RPPGFSPFR) acetate salt (98%) was purchased from Sigma (St Louis, MO, USA) and diluted to 2.5 μm in 50:49:1 (v/v/v) methanol water acetic acid. The peptide glycine leucine isoleucine serine histidine (GLISH) was synthesized with a CS036 peptide synthesizer from CS Bio (Menlo Park, CA, USA) and used without purification. The peptide GLISH was diluted to approximately 1 μm in 50:49:1 (v/v/v) methanol water acetic acid, and both bradykinin and GLISH samples were infused for electrospray ionization at 2 μl/min. All experiments were performed with a Bruker HCTultra ion trap mass spectrometer. The voltage applied to both the skimmer and the capillary exit was 1 V to reduce the amount of fragmentation that occurred within the instrument ion optics. Tandem mass spectrometry experiments were performed with the low mass cutoff set to 20% of the parent mass-to-charge ratio and an isolation width of 2.0. The electrospray ionization emitter was held at ground potential, and kv was applied to a previously described custom-built planar DIMS electrode assembly and housing [21]. The two parallel stainless steel electrodes (4 mm wide 10 mm long) are separated by a 0.3-mm gap. The assembly slides over the end of the instrument s resistive glass transfer capillary and is enclosed in a housing that reroutes the HCTultra desolvation gas (nitrogen) such that the desolvation gas also functions as the DIMS carrier gas. The actual temperature of the desolvation gas, and hence the DIMS carrier gas, is not as high as the set temperature because the temperature displayed by the mass spectrometer software is measured at the heating block rather than the inlet to the DIMS device. As the desolvation gas travels from the heating block to the inlet of the DIMS device, it transfers heat to the metal tubing and the DIMS device housing. This results in the temperature of the desolvation gas at the inlet to the DIMS device being lower than the temperature measured at the heating block. The flow rate of the desolvation gas also affects the temperature measured at the inlet to the DIMS device. A higher desolvation gas flow rate causes a greater amount of heat to be conducted to the gas transfer tubing and the DIMS device. Thus, the desolvation gas, transfer tubing, and DIMS device come to equilibrium at higher temperatures. The effect of this is that the temperature of the DIMS carrier gas is higher when greater desolvation gas flow rates are used at a given set temperature in the instrument software. The temperatures measured at the inlet to the DIMS device for each combination of temperature setting and desolvation gas flow rate used are listed in Table 1. Experiments with a 100% nitrogen carrier gas, a flow rate of 5.0 L/min, and a temperature setting of either 100 or 200 C used the mass spectrometer desolvation gas routed in the standard instrument configuration. For the experiments using 100% nitrogen carrier gas, a flow rate of 5.0 L/min, and a temperature setting of 300 C, the instrument desolvation gas tubing was disconnected before the heater and replaced with the output of a MKS model 1179 mass flow controller. For experiments with a mixed desolvation gas composition, the instrument desolvation gas tubing was again replaced, and the flow rate and composition were controlled through the MKS model 1179 mass flow controller and an Alicat MC-10SLPM-D mass flow controller. The heat transfer to the DIMS device was dependent on the gas composition, and the temperatures measured at the inlet to the DIMS device for each composition are also listed in Table 1. The dispersion field is generated with a bisinusoidal waveform formed by the capacitive coupling of two sinusoidal waveforms across the DIMS gap. The sinusoidal waveforms have frequencies of 2 and 4 MHz and are phase shifted by approximately 90. The waveforms are generated in an amplitude ratio of 2:1 (2 MHz/4 MHz), giving the waveform a form parameter of 0.67 [22]. DIMS spectra were generated as previously reported by our scanning the E C using a LabVIEW program linked to the instrument control software [8, 21, 23, 24]. After every ten mass spectra recorded by the instrument, E C was increased by 4 V/cm. Signal intensities and peak widths were calculated by our fitting the peaks in the DIMS spectra using MicroCal Origin 6.0 under the assumption of Gaussian Table 1. The temperature measured at the entrance to the differential ion mobility spectrometry device for each desolvation gas flow, temperature setting, and gas composition Gas composition Gas flow rate (L/min) Temperature setting ( C) Temperature measured ( C) 100% nitrogen ± % nitrogen ± % nitrogen ± % helium ± % argon ± % argon ± % carbon monoxide ± % carbon monoxide ± % carbon monoxide ± 0.8 Measurements were taken with a Klein Tools MM400 multimeter and a bead wire type K temperature probe.

4 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition 2163 distributions. MicroCal Origin 6.0 was also used to fit plots with sigmoidal curves and create the derivative plots of the fits. Results and Discussion Intra-DIMS Fragmentation of Peptides During DIMS analyses of bradykinin, the signal intensity for the [M + 3H] (m/z 354) ion decreased more much rapidly as a function of E D than that of the [M + 2H] (m/z 531) ion. This is depicted in Figure 2a. A decrease in signal as E D is increased is expected as the effective analytical gap is narrowed and it becomes likelier that ions will strike an electrode and be neutralized [25]; however, the more rapid fall off in signal intensity above E D = 29.2 kv/cm for the [M + 3H] ions compared with that of the [M + 2H] ions was interesting. Although not unprecedented because of differences in charge and possibly conformation, this behavior was further investigated to better understand how E D might affect ion 6 ) (x10 Intensity Signal 5 ) (x10 Intensity Signal 3.5 (a) [M+3H] +3 [M+3H-18] +3 (x5) [M+2H] +2 (x5) (b) Dispersion Field (kv/cm) [M+3H] [M+3H-18] Compensation Field (V/cm) Figure 2. a Signal intensity versus the applied dispersion field. b Compensation field scan performed at E D =33.3kV/cm transmission through the DIMS device. It was observed that as the signal intensity for the [M + 3H] ions decreased sharply as a function of E D, peaks corresponding to other mass-to-charge ratios increased in intensity as a function of E D. The most prominent of the peaks observed to increase in intensity as a function of E D during the analysis of bradykinin ions corresponds to ions of m/z 348, the signal intensity of which is also shown in Figure 2a. It should be noted from this figure that at all E D some ions of m/z 348 are detected; however, at the lower E D shown, these ions are due to the fragmentation of [M + 3H] ions within the ion optics of the mass spectrometer and are detected at the same E C as the [M + 3H] ions. An example E C scan at E D = 20.8 kv/cm is shown in Fig. S1, including traces of the signal intensity for the mass-tocharge ratio of both the [M + 3H] ion and the fragment ion. The fragment ions are detected at the same E C as the parent ions because the ions travel through the DIMS device as the parent and fragmentation occurs after the device in the mass spectrometer ion optics. As E D is raised and signal increases for m/z 348, the characteristic E C for the peak detected for m/z 348 shifts to a value slightly higher than that of the [M + 3H] ions. This small shift in E C is shown in Figure 2b. The corresponding decrease in signal intensity for the [M + 3H] ions and increase in signal intensity for the ions of m/z 348, along with the close proximity in terms of both mass-to-charge ratio and differential ion mobility, suggested that ions of m/z 348 were related to the [M + 3H] ions. To further substantiate the relationship, CID was performed on the [M + 3H] ions, and the resulting product-ion spectrum is shown in Fig. S2. Although significantly higher voltages are used in DIMS than during collisional activation in an ion trap, the higher pressures in DIMS devices shorten the mean free path, so the acceleration between collisions is limited. This causes the internal energy gained per collision to be small and for the heating to be similar to the heating that occurs during resonant excitation in an ion trap. The net result of this is that fragmentation proceeds via the lowest-energy pathway. During both CID and intra-dims fragmentation of the [M + 3H] ions of bradykinin, the primary fragment ion observed was of m/z 348, and is formed via a loss of neutral water. In both experiments, peaks were also observed for ions of m/z 322 (b 6 ), 419 (y 3 + ), 506 (y 4 + ), and 555 (b 5 + ). During intra-dims fragmentation at E D = 35.4 kv/cm and CID with a fragmentation amplitude of 0.33 the signal intensity ratios for the m/z 348 and m/z 354 ions were 4.87 and 4.25, respectively, meaning similar amounts of energy were added to the [M + 3H] ions. At those settings for intra-dims fragmentation, each of the less abundant fragment ions were detected at a relative signal intensity of less than 7% compared with the m/z 348 ion. During CID at the previously mentioned settings, the relative signal intensity for the m/z 322 ion (b 6 ) was 21%, with the relative signal intensity for the rest of the ions being below 10%. During DIMS scans, the intensity of the peaks for the ions formed via the less favorable pathways was increased in E C scans at higher E D ; however, the dissimilarities between the differential ion mobility of the fragment ions and the parent

5 2164 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition ions have the potential to cause significant disparities in ion transmission. These differences in ion transmission complicate the comparison of signal intensities. In general, the greater the disparity in differential ion mobility between the fragment ion and parent ion, the lower ion transmission would be expected to be for the fragment ion. During the intra-dims fragmentation of the [M + 3H] ions via the neutral loss of water, the fragment ions detected retain all three charges and are expected to have a structure similar to that of the [M + 3H] ions. As a result, the [M + 3H] ions and the water loss fragment ions have similar differential ion mobilities, with their characteristic E C separated by only 6.0 ± 0.7 V/cm at E D of 33.3 kv/cm. Therefore, it is expected that ion transmission for the [M + 3H] ions and that for the ions formed by the intra-dims loss of water would be similar, and signal intensities could be compared without our attempting to correct for ion transmission. A similar intra-dims fragmentation phenomenon is observed with the peptide GLISH, wherein y 3 + ions formed by intra-dims fragmentation passed through the DIMS device at an E C just slightly shifted from that of [M + H] +.Anexample E C scan is shown in Figure 3a, andtheincreaseiny 3 + signal intensity as a function of E D is displayed in Figure 3b. The close proximity in terms of E C for the [M + H] + ion and the y 3 + ion formed by intra-dims fragmentation leads to the belief that [M + H] + ions underwent fragmentation during transit through the DIMS device. However, CID experiments performed on the [M + H] + ions revealed that the formation of the b 5 + ion was the most favorable dissociation pathway (Fig. S3), yet no significant peak for the b 5 + ion was observed to appear as a function of E D in E C scans. Given that b 5 + corresponds to the water loss peak from [M + H] + and thus analogously to the bradykinin example earlier, it would be expected to observe this ion as an intra-dims fragment if the [M + H] + ion undergoes any fragmentation. Alternatively, in Fig. S4 it is shown that the most prominent product from CID of the [M + 2H] ion is the y 3 + ion. The CID spectra of the [M + H] + and [M + 2H] ions of GLISH, combined with the similar fragmentation patterns observed for CID and intra-dims fragmentation of the [M+3H] ions of bradykinin, allow the conclusion that the y 3 + ions formed via intra-dims fragmentation originate from [M + 2H] ions of GLISH rather than [M + H] + ions. The y 3 + ion is presumably detected at an E C closer to that of the [M + H] + ion of GLISH than the [M + 2H] ion, as shown in Fig. S5, because of the role charge state plays in determining the differential mobility of an ion. This lower charge state might also account for the difference in transmission observed between the fragment ions formed from bradykinin [M + 3H] and those formed from GLISH [M +2H]. If the signal intensity for bradykinin [M + 3H] ions that pass through the DIMS device at E D =16.7kV/cmis defined as 100%, only 16.8% of the bradykinin [M + 3H] ions are detected as the [M + 3H 18] ion when E D =33.3 kv/cm. If the same assumption about signal intensity is made for GLISH [M + 2H] ions at E D = 16.7 kv/cm, 30.8% of the ions are detected as y 3 + ions when E D = 33.3 kv/cm is applied. 6 ) (x10 Intenstiy Signal 6 ) (x10 Intensity Signal 1.4 (a) [M+H] + intra-dims y Compensation Field (V/cm) 4.0 (b) [M+2H] [M+H] + intra-dims y Dispersion Field (kv/cm) Figure 3. a Compensation field scan performed at E D =33.3 kv/cm. b Signal intensity versus the applied dispersion field. At E D = 16.7 kv/cm signal from y ions formed via intra-dims fragmentation cannot be distinguished from that of y + 3 ions formed by the fragmentation of [M + 2H] ions in the mass spectrometer ion optics. The signal intensity for y + 3 formed via intra-dims fragmentation was assumed to be zero at this E D The lower charge state of the y 3 + ion decreases the differential mobility of the ions, making it less likely they will strike one of the electrodes and be neutralized, and therefore increases transmission. The bradykinin [M + 3H 18] ions retain all three charges after intra-dims fragmentation, and thus the transmission is not affected in the same way as it is for GLISH. For both bradykinin and GLISH, only the more highly charged peptide species undergo intra-dims fragmentation. Neither the [M + 2H] ions of bradykinin nor the [M + H] + ions of GLISH were observed to fragment during transit through the DIMS device. This is particularly interesting for the [M + 2H] and [M + 3H] ions of bradykinin as they have nearly identical critical energies for the lowest-energy dissociations [26]. The slower drop in signal intensity as a function of E D for the [M + 2H] ions of bradykinin and the fact that no fragmentation is observed to occur for the [M + 2H] ions despite such similar internal energies being required for

6 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition 2165 dissociation highlight the greater acceleration experienced by the higher charge state ions as a critical factor for the addition of internal energy. Effect of Temperature on Fragmentation To further interrogate the intra-dims fragmentation of bradykinin [M + 3H] ions, the temperature setting of the mass spectrometer desolvation gas, and therefore the DIMS carrier gas, was varied. The principle of this work was based on studies where elevating the bath gas temperature during CID in a quadrupole ion trap was shown to increase the initial internal energy of the ions before collisional activation and facilitate fragmentation [27]. In the current work, the ions were thermalized with the carrier gas, and therefore the temperature of the carrier gas determines the initial internal energy of the ions. This gives the internal energy of the bradykinin ions initially to be 14.24, 15.87, and ev at carrier gas temperatures of 91.7, 133.4, and C, respectively, with use of U ¼ U T þ U R þ U V ð1þ where U is the total internal energy of the ion, U T is the amount of internal energy supplied by translational motion, U R is the internal energy added by rotational energy, and U V is the vibrational contribution to internal energy. From these internal energy calculations, as the temperature of the carrier gas is increased, less internal energy would need to be added via collisional heating for the ions to reach the internal energy required for intra-dims fragmentation to occur. This effect is shown in Figure 4a, where the fragmentation efficiency of the [M + 3H] ion to the [M + 3H 18] ion is plotted. As the temperature of the carrier gas is increased, lower dispersion field strengths are required for the appearance of [M + 3H 18] ions. From fitting of the values shown in Figure 4a obtained at carrier gas temperatures of and C with sigmoidal curves, it can be determined that the 0.85-eV difference in initial internal energy requires an E D 3.3 kv higher for the ions to reach an internal energy where the fragmentation efficiency of the [M + 3H] ions is 50%. With use of Origin to extrapolate the data measured at 91.7 C, the plot show in Figure 4b was generated. This shows a linear relationship between the initial internal energy of the [M + 3H] ions and the E D at which the fragmentation efficiency reaches 50%. The same experiment was also performed with the [M + 2H] ions of GLISH, which were calculated to have internal energies of 7.26, 8.09, and 8.53 ev at carrier gas temperatures of 91.7, 133.4, and C, respectively, with use of Eq. 1. The data for the y 3 + fragment ion from GLISH [M + 2H] are shown in Figure 5. As with the bradykinin [M + 3H 18] ions, the GLISH ions with higher initial internal energies require that less internal energy be added by collisional heating for intra-dims fragmentation to occur; therefore, the fragment ion is detected at lower E D. Fitting the data in Figure 5a with sigmoidal curves yields the E D at which the fragmentation Efficiency Fragmentation E D at Fragmentation Efficiency = 50% (kv/cm ) 1.0 (a) Dispersion Field (kv/cm) (b) 91.7 o C o C o C Linear Fit y= -4007x R 2 = Initial Internal Energy (ev) Figure 4. a Dispersion field versus fragmentation efficiency at different carrier gas temperatures to determine the relationship between E D and internal energy deposition for the [M + 3H] ions of bradykinin. b E D where 50% fragmentation efficiency for the bradykinin [M + 3H] ions is reached versus the initial internal energy efficiency of the [M + 2H] ions reaches 50%. These values are plotted versus the initial internal energy of the [M + 2H] ions in Figure 5b. As with bradykinin, the relationship was found to be linear. Thus, the difference in E D where the fragmentation efficiency of the parent ions reaches 50% can be converted to a difference in the amount of internal energy gained during transit through the DIMS device. A comparison of the slopes in Figures 4b and 5b shows that as E D is increased, the effect on the internal energy of the [M + 3H] ions of bradykinin is greater than that on the [M + 2H] ions of GLISH. Although bradykinin has significantly more degrees of freedom over which to spread the energy, the higher charge state of the ion could account for this greater internal energy increase as E D is increased. Changing the temperature of the DIMS carrier gas to manipulate the internal energy of ions during an analysis by DIMS

7 2166 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition Efficiency Fragmentation E D at Fragmentation Efficiency = 50% (kv/cm ) (a) 91.7 o C o C o C Dispersion Field (kv/cm) (b) Linear Fit y= -8853x R 2 = Initial Internal Energy (ev) Figure 5. a Dispersion field versus fragmentation efficiency at different carrier gas temperatures to determine the relationship between E D and internal energy deposition for the [M + 2H] ions of GLISH. b E D where 50% fragmentation efficiency for the GLISH [M + 2H] ions is reached versus the initial internal energy. (Note the error bars are too small to be visible on the scale of the plot.) must be done in balance with achieving the separation required. Decreasing the carrier gas/ion temperature could be valuable for compounds with low isomerization/fragmentation barriers, at the risk of limiting separation abilities. Changes to the temperature of the DIMS carrier gas alter the number density (N) within the DIMS device, and because ion mobilities are dependent on E/N, variation of the temperature modifies the separation characteristics [28, 29]. For the ions formed by water loss from the [M+3H] ions used in this work, the overall separation ability was observed to be directly related to the temperature of the DIMS carrier gas. The characteristic E C and width of the peaks are shown in Figs. S6 and S7, respectively, for all temperature and E D settings used. It was observed that hotter DIMS carrier gas temperatures lead to higher E C, and thus the potential separation power of DIMS for these ions is highest at the same settings that facilitate fragmentation. This improvement in separation ability at higher carrier gas temperatures applies to all peptide ions examined by DIMS, as they have exhibited differential mobilities that increase as E/N is raised [2]. Therefore, increasing the temperature of the carrier gas will increase the characteristic E C of peptide ions and improve the separation ability of the DIMS device for these species but will also increase the likelihood of intra-dims fragmentation. Hence, the potential trade-off between separation capability and the retention of structural information must be considered on the basis of the experimental circumstances. Effect of Carrier Gas Composition on Fragmentation Another method that has been shown to change the amount of internal energy deposited into ions during DIMS analyses is to vary the composition of the carrier gas with use of nonpolar gases [4, 16]. The change in internal energy of an ion due to collisional heating in a DIMS device, measured as the change in the temperature of the ion (ΔT), has been shown to depend on the inverse square of the collision cross section (Ω)byEq.2: 3π ΔT ¼ 128k 2 B T 1 þ M zee 2 ; ð2þ m NΩ where k B is the Boltzmann constant, T is the temperature of the carrier gas, M is the mass of the carrier gas, m is the mass of the ion, z is the ion charge state, e is the elementary charge, E is the electric field strength, and N is the number density of the gas [4, 29]. Thus, use of carrier gases other than nitrogen would affect the amount of internal energy deposited. The effect on intra- DIMS fragmentation of changing the carrier gas composition to be a mixture of nitrogen and helium, argon, or carbon monoxide is shown in Fig. S8. Similarly to Figures 4a and 5a, Fig. S8 shows the fragmentation efficiency of the bradykinin [M + 3H] ion versus the applied E D. Although the differences are difficult to observe from Fig. S8, a plot of the derivative of the sigmoidal fits to the data as shown in Figure 6 more clearly shows differences in the relationship between E D and intra- DIMS fragmentation with different carrier gas compositions. Although Figure 6 shows these changes in a qualitative way, the data obtained during experiments varying the carrier gas temperature allow a more quantitative approach to comparing internal energy deposition by a nitrogen carrier gas versus a gas mixture. Comparison of Fig. S8a, which was obtained with a 100% nitrogen carrier gas, and Fig. S8b, which was obtained with a 20% helium 80% nitrogen mixture, shows that the E D needed for intra-dims fragmentation to occur when helium is added to the carrier gas is significantly lower. When the DIMS carrier gas is 20% helium, the E D where 50% fragmentation efficiency is reached is 1.9 kv/cm lower compared with that for a 100% nitrogen carrier gas. From the relationship shown in Figure 4b, a decrease of 1.9 kv/cm in

8 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition ) (x10 Fit Efficiency Fragmentation of Dispersion Field (kv/cm) Figure 6. Slope of the sigmoidal curves fit to the data in Fig. S8 versus the applied dispersion field Slope 100% Nitrogen 20% Helium 20% Argon 40% Argon 20% Carbon Monoxide 40% Carbon Monoxide 60% Carbon Monoxide the E D where the fragmentation efficiency reaches 50% is equivalent to an increase in internal energy of 0.35 ev. However, the ions also start with lower internal energies when 20% helium is used because of the difference in heat transfer between the carrier gases. The amount of internal energy gained by the ions in a 20% helium carrier gas compared with a 100% nitrogen carrier gas must also include the 0.29-eV difference in initial internal energy. Therefore, at the E D where the fragmentation efficiency reaches 50% when 20% helium is used (E D = 31.2 kv/cm), the DIMS carrier gas including helium has added 0.64 ev more internal energy to the ions. As with the addition of helium to the carrier gas, the use of 20% and 40% argon (Figs. S8c and S9d, respectively) resulted in more internal energy deposition into the bradykinin [M + 3H] ions than with a 100% nitrogen carrier gas. Use of 20% argon causes the E D where a 50% fragmentation efficiency is reached to be 1.0 kv/cm lower than when a 100% nitrogen carrier gas is used. That 1.0 kv/cm difference is the equivalent of the ions being 0.19 ev higher in internal energy. As with helium, carrier gas mixtures including argon are cooler than when 100% nitrogen is used. Therefore, the difference in initial internal energy must be included to determine how much more internal energy has been added by the carrier gas including argon. Thus, the 0.19 ev must be combined with the 9-eV difference in initial internal energy, meaning that at E D = 32.1 kv/cm, the carrier gas including 20% argon has increased the internal energy of the ions by 0.28 ev more than the 100% nitrogen carrier gas. The use of 40% argon in the carrier gas lowers the E D required for a fragmentation efficiency of 50% by 2.2 kv/cm, equating to the ions having an internal energy 0.41 ev higher. The 40% argon carrier gas is also significantly cooler than the 100% nitrogen carrier gas, and the initial internal energy of the ions is 0.20 ev lower. From these values, at an E D of 30.9 kv/cm, the use of 40% argon carrier gas has raised the internal energy of the ions 0.61 ev more than when 100% nitrogen is used. Had the temperature difference between the gas mixtures and 100% nitrogen not been present, the more efficient internal energy deposition that occurs when helium and argon are used would have caused fragmentation at even lower E D. Also worth noting is that the amount of internal energy increase relative to 100% nitrogen when 20% helium or 40% argon is used is nearly the same (0.64 ev vs 0.61 ev). This highlights the importance of collision cross section on the change in internal energy as ions pass through a DIMS device. Although argon has a mass nearly ten times that of helium, which increases the maximum amount of center-of-mass collision energy available by a factor of almost 10, the heavier argon atoms increase the internal energy of the ions less than the lighter helium atoms. This is believed to occur because argon has a greater collision cross section with the ions [30]. This results in more frequent collisions and thus lower terminal velocity between collisions, leading to lower collision energies. This is contrary to what is observed when gases other than helium are used as the collision gas in an ion trap, where heavier gases cause more fragmentation than helium [31, 32]. This can be rationalized on the basis of the collision frequency, which is much lower in the ion trap, and thus a greater terminal velocity can be reached between collisions. Because of the higher kinetic energy of the ions in the ion trap, the collision cross sections will be smaller and there will be smaller differences in collision cross section between gases. This results in the collision gas mass, and therefore the available center-ofmass collision energy, being the dominant variable for the addition of internal energy in an ion trap. Changing the carrier gas composition can also lower the amount of internal energy added as an ion travels through a DIMS device. Use of a more polar gas such as carbon monoxide increases the collisional cross section, and the internal energy deposition can be lowered [33]. Gas mixtures containing 20%, 40%, and 60% carbon monoxide result in significantly less intra- DIMS fragmentation, the data for which are presented in Fig. S8e g. A plot of the percentage of carbon monoxide present in the carrier gas versus the E D at which the fragmentation efficiency of the [M + 3H] ions of bradykinin reaches 50% yields the linear relationship shown in Figure 7, and the nonzero slope of the plot confirms that collisional cross section plays a role in the internal energy deposition process. The same calculations that were performed for helium and argon carrier gas mixtures to describe the difference in internal energy added with different carrier gas compositions can be done for carrier gas mixtures with carbon monoxide. However, carrier gas mixtures including carbon monoxide add less internal energy compared with a 100% nitrogen carrier gas. When 20%, 40%, and 60% carbon monoxide mixtures are used as the DIMS carrier gas and E D = 33.1 kv/cm is applied, the internal energies of the ions are 0.12, 0.28, and 0.48 ev lower, respectively, compared with when a 100% nitrogen carrier gas is used. These values include the slight differences in initial internal energies based on gas temperature. From the results obtained it can also be concluded that changing the composition of the carrier gas has similar implications on separation power as manipulating the carrier gas temperature.

9 2168 B. G. Santiago et al.: Intra-DIMS Internal Energy Deposition E D at Fragmentation Efficiency = 50% (kv/cm ) % Carbon Monoxide in Carrier Gas Figure 7. E D where 50% fragmentation efficiency for the bradykinin [M + 3H] ions is reached versus the percentage of the carrier gas that is carbon monoxide Although addition of helium increases the difference between low-field and high-field ion mobilities (data shown in Fig. S9) and has been shown to improve separations, it also increases the amount of internal energy deposited into an ion at any E D [4, 8, 34, 35]. Furthermore, helium has a considerably lower breakdown voltage than nitrogen at the pressures and distances typically used in DIMS, placing an upper limit on the E D that can be used [8, 36]. The use of higher amounts of carbon monoxide reduces the amount of internal energy ions gain during their transit through a DIMS device, but also potentially reduces the separation power of the analysis [34, 35]. For the [M + 3H] ions in this work, the use of carbon monoxide lowers the characteristic E C, whereas peak widths remain constant (data shown in Fig. S10). These effects will be compound-specific on the basis of the interactions of the ion and the carrier gas, but for peptides the use of carbon monoxide will lower the characteristic E C of the ions and therefore reduce the separation ability of the DIMS device [29]. Thus, the trade-offs must be considered on the basis of the experimental requirements and limitations. Conclusions Linear Fit y= x R 2 = The intra-dims fragmentation of peptides has been observed, and the fragmentation patterns strongly correlate with those of CID performed in an ion trap mass spectrometer. Thus, it is believed that the collisions that occur within a DIMS device add energy via a slow heating process that favors fragmentation proceeding via the lowest-energy pathway. However, the energy deposition process is different between ion trap CID and collisions in the DIMS device. For the former, increasing collision gas mass increases internal energy deposition, whereas for the latter, the energy deposition is inversely related to the collision cross section (i.e., gases with higher cross sections deposit less internal energy). The effect of varying the DIMS carrier gas temperature, and therefore the ion internal energy before collisional heating, was studied. The E D amplitude at which the fragmentation efficiency of the parent ion reached 50% was found to have a linear relationship with the initial internal energy of the ions. Thus, over the E D range studied, internal energy increased in a linear fashion with the applied E D. The internal energy deposition was also found to be more efficient for the [M + 3H] ions of bradykinin versus the [M + 2H] ions of GLISH, which is believed to occur because of the higher charge state of the bradykinin ions studied. It was also shown that by our varying the composition of the DIMS carrier gas the amount of internal energy deposited into ions could be adjusted. Gases such as helium and argon yielded greater internal energy deposition than the typically used nitrogen carrier gas, whereas the more polar carbon monoxide has a greater collision cross section with ions and the amount of internal energy gained by the ions is lower when they pass through a DIMS device with carbon monoxide in the carrier gas. The difference in internal energy deposited when different carrier gas compositions were used compared with a 100% nitrogen carrier gas was calculated from the relationship between E D and the internal energy of the ions determined while the temperature of the carrier gas was varied. The relationship between the amount of carbon monoxide present in the carrier gas and the E D required for the fragmentation efficiency of the parent ion to reach 50% was also found to be linear, with a positive correlation, confirming that collision cross section plays a role in the internal energy deposition process. Thus, both varying the carrier gas temperature and altering the carrier gas composition yield the ability to tune the amount of internal energy deposited into ions during their transit through the DIMS device. 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