Introduction

The physiological properties of proteins, peptides, high-molecular weight compounds and other biologically active compounds are strongly related to their structures. Hence, structure determination of the aforementioned compounds are pertinent. Mass spectrometry (MS) based analytical techniques have widely been used to analyze and characterize peptides,[1, 2] proteins,[3, 4][1, 5], [3, 4],[1, 5] and other types of molecules including isotopes[6,7,8]and isotopologues[9,10,11] with high sensitivity. However, as a technique which measures the mass-to-charge ratio of ions, MS suffers from the inability to separate gaseous isomers and conformers.[12,13,14] Ion mobility spectrometry (IMS) finds growing utility in efforts to probe the shape and conformation of small molecules and proteins in the gas-phase by separating ions based on their gas-phase ion mobilities which is conceptually linked to the concept of a charge-normalized collision cross section.[15, 16] When combined with mass spectrometry, IM-MS offers a tractable path to achieve isomeric separations by providing structural information of molecules of interest since the structural separation obtained by IMS is uniquely different from mass-to-charge information. The application of IM-MS as analytical technique has evolved to include a broad range of research disciplines from structural biology analysis,[17] drug metabolism,[18] native protein-protein assemblies,[19] isomeric separations,[20,21,22] and molecular class differentiation.[23, 24].

Despite the broad applicability and increasing usage, ion mobility is a relatively low resolution separation technique. Signal average measurements made with traditional drift tube systems suffer from poor sensitivity due to its inherent low duty cycle related to the period between ion gating cycles and the initial width of the ion packets injected into the drift tubes. Multiplexing techniques based on ion-gate modulation, such as Hadamard and Fourier-transform, are known to provide a solution to the low duty cycles observed in signal averaged drift tube systems.[25,26,27] Fourier-transform based ion gate modulations have made it possible to couple IMS experiments to mass analyzers with lower sampling rates such ion traps,[26, 28, 29] and obitraps,[30] with a 25-fold enhancement in duty cycle compared to signal averaged ion mobility experiments. Some isomeric species require higher resolution in the ion mobility spectrometer to achieve separation.[22] Therefore, for IM-MS techniques that use linear frequency modulation as a mobility encoding scheme, higher resolution separations are accessed through efficient ion gating and higher frequencies.[28] However, ion gate depletion effects (widely described in Bradbury-Neilson (BN) ion shutters)[25, 31, 32] are known to severely impact slower moving ions (i.e. lower mobilities) and are exacerbated at higher ion gating frequencies.[21] Regardless of the ion transmission mechanisms used in either high pressure or low pressure drift tube IMS experiments, mobility bias exists. For low-pressure drift tube IMS systems using ion funnel traps as the means of ion injection, efforts to reduce ion discrimination by employing helium as a trapping gas have shown promise, however, this work conducted by Ibrahim et al. requires a careful balance of differential pressures and is not applicable to drift tube IMS systems with pressures much above 10 Torr.[33].

Recently, Kirk et al. described an improved configuration (known as tri-state, tri-grid ion shutter (3S-IS)) [34] based upon the previous tri-grid ion shutter (two-state ion shutter (2S-IS))[35] which showed a significant improvement over the signal averaged mode experiments with a major decrease in ion depletion effect for a gate pulse width of 1 μs.[34] In our recent publication,[21] a comparison of the performance levels of the 3S-IS to 2S-IS were made for a series of Fourier-transform ion mobility mass spectrometry experiments. The 3S-IS and 2S-IS comparison were made using compounds with a range of reduced mobilities such as peptides and a series of tetraalkylammonium salts (T5-T8, T10 and T12 ions). Increases in ion counts of 95% and 45% for T5 and T12 ions respectively were reported with the 3S-IS compared to the 2S-IS. Overall a 10% increase in resolving power was achieved with the 3S-IS compared to the 2S-IS. Furthermore, from the same publication, we were able to perform isomeric separation for two reversed sequence pentapeptides using the 3S-IS. With a resolving power of 79 and 57 reported for SDGRG and GRGDS singly charged ions using 3S-IS, a clear discrimination between the two isomers were observed although a baseline resolution was not achieved for these two singly charged species.

Building upon the isomeric separation experiments made in our previous publication, the purpose of this present study is to perform ion mobility separations on different pairs of isomeric species using ion mobility coupled to a linear ion trap mass spectrometer with a Fourier-based ion gate modulation technique in combination with the 3S-IS and 2S-IS. A comparison of the ion mobility separations achieved using the 3S-IS and 2S-IS was investigated for a mixture of reversed sequence pentapeptides, hexapeptides, and heptapeptides.

Experimental

Chemicals and Reagents. The target analytes chosen were different pairs of isomeric peptides. These included: (1) Gly-Arg-Gly-Asp-Ser (GRGDS)/Ser-Asp-Gly-Arg-Gly (SDGRG);[21] (2) Sar-Arg-Gly-Asp-Ser-Pro (SRGDSP)/Gly-Arg-Gly-Asp-Thr-Pro (GRGTP);[22] (3) Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)/Leu-Arg-Arg-Ala-Ser-Val-Ala (Val6, Ala7-Kemptide).[22] In addition to the analytes listed above, HPLC grade methanol,water and 0.1% formic acid (ACS reagent grade, > = 97 or 98% purity) were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). These mixtures were infused into a custom electrospray ionization emitter consisting of a 75 μm glass capillary with the terminating polyimide coating removed. Each isomeric peptide pair was formulated at a concentration of 50 μM in 80/20 HPLC grade methanol / water with 0.1% formic acid respectively.

Atmospheric dual-gate Ion Mobility-Mass Spectrometry. The drift time measurements were made using an atmospheric pressure, PCB-dual ion shutter ion mobility spectrometer coupled to a linear ion trap mass spectrometer. The PCB-dual ion shutter IMS were modelled after the design described by Reinecke and Clowers.[36] The aforementioned dual ion shutter IMS was sealed by using teflon spacers tightly compressed between the electrodes used for both the desolvation region (10.02 cm) and the drift tube. The length of the drift tube was approximately 18.5 cm (with a length of 17.5 cm between the first and second gate). The mass-selected mobility spectra were obtained using the PCB-IMS coupled to a linear ion trap mass spectrometer,(LTQ-XL, Thermo Fisher Scientific, Thousand Oaks, CA) by frequency encoding the mobility data.[21] With the linear ion trap mass spectrometer as the detector, the ion trap was filled for a fixed amount of time by setting the target accumulation population to a large value as AGC could not be turned off in the standard LTQ-XL control software. With this known AGC setting in place, a maximum injection time of 100 ms and m/z ranges 50–500, 50–700, and 50–800 were used for the analysis of reversed sequence pentapeptide, hexapeptide, and heptapeptide respectively. The frequency modulated IMS experiments (Fourier Transform-IMS (FT-IMS)) were swept from 5 to 10,005 Hz over the course of 8 min. In addition to the experimental conditions listed above, the analytes were electrosprayed at a flow rate of 3 μL/min through a 75 μm glass capillary. An electrospray ionization voltage of 2500 V (relative to the first IMS electrode) was used. With the potential of the first IMS electrode held at 15000 V, an electric field of ~467 V cm−1 was realized. The IMS was operated at atmospheric pressure (~ 690 Torr in Pullman WA) and maintained at room temperature (23 °C). A countercurrent nitrogen gas flow of 2.5 L/min was allowed into the drift tube (This higher value of drift gas flow was used to account for the API inlet for the mass analyzer). The ions were gated using the two-state, tri-grid ion shutter (2S-IS)[35] and the tri-state, tri-grid ion shutter (3S-IS) principles with the latter first being introduced by Kirk et al.[34] and also described in great detail in our recent publication.[21] The first and second gate were operated with open source ion gate pulsers [37] triggered using a National Instruments Multifunctional DAQ (USB-6531, National Instruments, Austin, TX) that provided the waveforms needed for the experiments. The linearly swept waveforms were delivered to the ion gating electronics through the USB-6531 by leveraging the built-in tools found in the NIDAQ Tools module extension for IGOR Pro (Lake Oswego, OR). We note that a potential of 150 V was applied to completely close the gate in order to stop ions from entering the drift region. Structurally, the arrangement and assembly of the gates are in the same order as that reported by Kwantwi-Barima et al.[21].

Discussion

For all the peptides analyzed, both singly and doubly charged ions were observed in our FT-IMMS experiments. The reduced mobility (K0) and ion-neutral collision cross section (CCS) for each peptide ion reported in Table 1 were calculated from the individual analyte measurements and not the peptide mixture. Figure 1 is the mass selected mobility spectra for reversed sequence pentapeptide GRGDS and SDGRG obtained through FT-IMMS experiment from 5 to 10,005 Hz for 8 min. Figure 1a and b represent the singly and doubly protonated ions of isomeric pentapeptide mixture using the 2S-IS and 3S-IS respectively. Whereas, Fig. 1c depicts the singly and doubly charged ions of the individual pentapeptide target analytes. For the 3S-IS, baseline separation was achieved for both singly and doubly protonated ions of the isomeric pentapeptide mixture. However, for the 2S-IS, both singly and doubly charged ions of the pentapeptide mixture remained unresolved in the mobility domain as depicted in Fig. 1a. The separation of the singly and doubly protonated ions were due to the small differences in their conformational structure measurable as ion-neutral collision cross section (CCS). The CCS values calculated from the experimental measurements were 205 ± 0.42 Å2, 203 ± 0.43 Å2, 269 ± 0.89 Å2, 274 ± 0.87 Å2 for [GRGDS+H]+, [SDGRG+H]+, [GRGDS+2H]2+, and [SDGRG+2H]2+ respectively. The original measurements of GRGDS and SDGRG by Wu et al. were conducted using a high temperature drift cell with subsequent measurements made independently by Bush et al.[38], and May et al.[24] Recognizing that the conditions used by May et al. [24] are most similar to the present instrument (i.e. room temperature, nitrogen gas) the singly charged values (205.9 ± 0.2 Å2, 204.6 ± 0.5 Å2 for [GRGDS+H]+ and [SDGRG+H]+ respectively) agree within experimental error of 0.5%. Unfortunately, in that effort, data for the double charged species were not reported. Close examination of the reported literature for the doubly charged species highlights a gap in the consistency of measurement conditions which hinders direct comparisons. Placing the present values for the doubly charged species in the context of the elevated temperature work of Wu et al.,[22] (222.7 Å2, 211.7 Å2 for [SDGRG+2H]2+ and [GRGDS+2H]2+ respectively) and the radially confining drift cell by Bush et al.[38] (259 Å2, 256 Å2 for [SDGRG+2H]2+ and [GRGDS+2H]2+ respectively); which itself slightly elevates the effective temperature of the ion, the trends in our data agree with the notion of effective temperature in a radially confining cell (within an experimental error of 6%), and the non-linear dependence of mobility on temperature from a qualitative perspective. It is for these reasons that the values reported here for the double charged species require continued verification using different experimental systems and nominally the same environmental conditions. Furthermore, from the structure of the peptides, the charge location for the doubly protonated ions can reasonably be predicted from the N-terminal residue and the the basic arginine group (GlyH+-ArgH+-Gly-Asp-Ser; SerH+-Asp-Gly-ArgH+-Gly). From the CCS values listed in Table 1 for the doubly charged ions, the [SDGRG+2H]2+ seems to exhibit a more extended conformation where the charges are separated from each other (hence the larger CCS value of 259 Å2) stabilized by surrounding carbonyl groups than the [GRGDS+2H]2+ which may exhibit a more compact conformation (smaller CCS value of 256 Å2) due to the location of the charges and hence presumably providing more coulombic repulsion as reported by Wu et al.[22] In Table 2, the ion counts recorded for the isomeric pentapeptide mixture at m/z 246 and m/z 491 with the 3S-IS were 9.91 × 103 and 2.25 × 104 corresponding to a 93.3% and 84.0% increase for m/z 246 and m/z 491 respectively in comparison to the 2S-IS. The resolving powers for the reversed sequence pentapeptide mixture with the 3S-IS were 100 and 115 for [GRGDS+H]+, [SDGRG+H]+ respectively and 128, 82 for [GRGDS+2H]2+ and [SDGRG+2H]2+ respectively. The resolving powers for the individual species were 104 for both [GRGDS+H]+, [SDGRG+H]+ and 49, 50 for [GRGDS+2H]2+ and [SDGRG+2H]2+ respectively. Under the reported conditions, baseline resolution of both singly and doubly charged ions of the two isomeric pentapeptide mixture was attainable with PCB-IMS system, illustrating that the resolving power of the present instrument with the 3S-IS setup allows separation for isomeric compounds with smaller differences in CCS values (< 1%). We note that, this is the first time baseline separation has been reported for the singly charged ions of the isomeric reversed sequence pentapeptide mixture using Fourier transformed drift tube IMS with nitrogen as the drift gas.

Table 1 Summary of molecular weight, m/z, reduced mobilities and Collision cross section (CCS)
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Fig. 1
figure 1

Mass selected mobility spectra obtained through the FT-IMMS experiment with a frequency sweep from 5 to 10,005 Hz for 8 min using the (a) two-state ion shutter (2S-IS) for a mixture of two reversed sequence pentapeptide, (b) tri-state ion shutter (3S-IS) for a mixture of two reversed sequence pentapeptide. (c) The two pentapeptides were analyzed individually using the 3S-IS

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Table 2 Summary of ion counts for the target analyte ion mixture using the 3S-IS and 2S-IS
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The primary difference between the two isomeric hexapeptides seen in Fig. 2 is the location of the methyl group. The methyl group on the sarcosine amino group is shifted to the serine thereby converting the sarcosine to glycine and the serine to threonine. Figure 2a and b are the mass selected mobility spectra of isomeric hexapeptides ion mixture obtained with the 3S-IS and 2S-IS respectively. Whereas, Fig. 2c displays the mass selected mobility spectra of individual hexapeptides target analyte ions. According to Table 1, the CCS value of the singly protonated ions were experimentally the same with a difference of 0.4%, significantly smaller than the doubly protonated ion population and remained unseparated. However, a baseline separation was achieved for the doubly protonated ions with the 3S-IS as shown in Fig. 2b. Whereas, with the 2S-IS, a near-to-baseline separation was achieved for the doubly charged ions as seen in Fig. 2a The measurement indicated a difference of 1.0% in the CCS value calculated for the doubly protonated ion. In comparison to the work done by Wu et al.,[22] a difference of 2.3% was recorded for the doubly charged ion species, which resulted in a near-to-baseline separation. This results further demonstrates the improved performance of the 3S-IS to separate ions with significantly smaller differences in their calculated CCS values and also has a higher resolving power than the resolving power of the IMS reported by Wu et al. (RP = 80) [22]. Additionally, the ion counts and resolving power of the hexapeptide ion mixture with the 3S-IS was higher than that of the 2S-IS. For the hexapeptide mixture at m/z 602 and m/z 301, the recorded ion counts in Table 2 with the 3S-IS were 3.18 × 104 and 5.56 × 104 resulting in a 61.9% and 88.4% increase for m/z 602 and m/z 301 respectively in comparison to the 2S-IS. For reference, the resolving powers for the hexapeptide mixture with the 3S-IS were 87 and 104 for [GRGDTP+2H]2+, [SRGDSP+2H]2+ respectively and for the 2S-IS were 80 and 100 for [GRGDTP+2H]2+, [SRGDSP+2H]2+ respectively. This corresponds to an 8% and 3.8% increase in resolving power for [GRGDTP+2H]2+ and [SRGDSP+2H]2+ respectively with the 3S-IS in comparison to the 2S-IS. Whereas, the resolving powers recorded for the individual species were 57 for [GRGDTP+2H]2+ and 82 for [SRGDSP+2H]2+. The calculated CCS values for the doubly protonated ions of SRGDSP and GRGDTP were (278 Å2) and (281 Å2) respectively. SarH + -ArgH + −Gly-Asp-Ser-Pro, GlyH + -ArgH + −Gly-Asp-Thr-Pro are the suggested charge locations for the doubly protonated ions. The closeness of the two charges might presumably cause strong coulombic repulsion. The calculated CCS values suggest that [GRGDTP+2H]2+ exhibits a more elongated structure (“stretched out”) than [SRGDSP+2H]2+. The differences in the gas-phase conformation exhibited by the doubly protonated ions might be due to the differences in gas basicities demonstrated by the N-terminal groups (888.7 kJ/mol for sarcosine and 855.4 kJ/mol for glycine). The conformations of peptides and proteins following electrospraying ionization are impacted by a range of factors including solvent ionic strength, site(s) of protonation or deprotonation, and gas-phase basicity.[39,40,41,42] Regarding the latter property, sarcosine and glycine serve as salient comparisons when considering gas-phase basicity in the context of the present experiments. The differential proton affinity, which is directly related to gas-phase basicity, between the two amino acids establishes conditions where different degrees of Coulombic repulsion are present when the sites of protonation are at the two amino acids adjacent to the N-terminus. Under these conditions, in order to accommodate the higher coulombic repulsion between the sarcosine and arginine charge sites, conformational rearrangement is likely in the gas-phase which translates into a different gas-phase ion conformation for SarH + -ArgH + −Gly-Asp-Ser-Pro with a CCS lower than that of GlyH + -ArgH + −Gly-Asp-Thr-Pro. This rationale also extends to the hexapeptide systems where [GRGDTP+2H]2+ exhibits a more elongated structure than [SRGDSP+2H]2+. Building along this same line, Wu et al.[22] suggested that to overcome the strong Coulombic repulsion, the charge located on the glycine (GlyH + -ArgH + −Gly-Asp-Thr-Pro) might shift to the carbonyl side on the backbone of the sequence to produce a different gas-phase conformation.[43] The present discussion assumes the charge sites for the doubly charged species are restricted to the N-terminus, however, the possibility of alternative charge configurations cannot be entirely excluded. Figures 1 and 2 display Gaussian fits to the transformed FT-IMMS spectra, however, close examination of the raw traces illustrates that small shoulders exist for some of the doubly charged species. These observations further support the notion that alternative conformations can exist and the core aspects of the discussion regarding Coulombic repulsion hold.

Fig. 2
figure 2

Mass selected mobility spectra of two isomeric hexapeptide different by the N-terminal amino acids and the fourth amino acid obtained through the FT-IMMS experiment with a frequency sweep from 5 to 10,005 Hz for 8 min using the (a) two-state ion shutter (2S-IS) for the ion mixture, (b) tri-state ion shutter (3S-IS) for ion mixture. (c) The two hexapeptides were individually analyzed using the 3S-IS

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Kemptide and Val6,Ala7-kemptide were analyzed and observed as both singly ([kemptide+H]+, [(Val6, Ala7)-kemptide+H]+) and doubly protonated ([kemptide+2H]2+, ([Val6, Ala7)-kemptide+2H]2+) ions as illustrated in Fig. 3. Figure 3 is the mass selected mobility spectra for two heptapeptide ion mixture using 2S-IS (Fig. 3a) and 3S-IS (Fig. 3b). Whereas, Fig. 3c is the mass selected mobility spectra of the individual heptapeptide analyte ions. The difference between the two heptapeptides is the shift of a methyl group from the leucine to glycine; converting leucine to valine and glycine to alanine. The possible charge locations for both kemptide and (Val6, Ala7)-kemptide singly charged ions are as follows. 1) LeuH+-Arg-Arg-Ala-Ser-Leu-Gly / (Val6, Ala7), 2) Leu-ArgH+-Arg-Ala-Ser-Leu-Gly / (Val6, Ala7), 3) Leu-Arg-ArgH+-Ala-Ser-Leu-Gly / (Val6, Ala7). From Fig. 3a–c, a single ion mobility peak was observed for [kemptide+H]+. If all the proposed charge locations for [kemptide+H]+ had been produced during the electrospray process then three ion populations would be present in the system. While baseline separation of these protomers may not be possible using the present system, a modicum of peak broadening would be expected under such conditions. Instead, a single ion mobility peak was observed for [kemptide+H]+. However, for [(Val6, Ala7)-kemptide+H]+, two ion mobility peaks were observed in Fig. 3a–c. The two ion mobility peaks for the Val6, Ala7-kemptide singly charged ion demonstrates that two of the listed proposed charge location configurations were captured in the gas-phase when Val6, Ala7-kemptide was electrosprayed. The CCS values calculated from the individual target analyte ions for the singly charged ions were 276 Å2 for kemptide and 268 Å2, 279 Å2 for Val6, Ala7-kemptide as observed in Table 1. These CCS values correspond to differences of 3.0% and 1.1% which resulted in the baseline separation as shown in Fig. 3a and b using the 2S-IS and 3S-IS respectively. For the doubly protonated species, the suggested charge locations are as follows. 1) LeuH+-ArgH+-Arg-Ala-Ser-Leu-Gly / (Val6, Ala7), 2) LeuH+-Arg-ArgH+-Ala-Ser-Leu-Gly / (Val6, Ala7), 3) Leu-ArgH+-ArgH+-Ala-Ser-Leu-Gly / (Val6, Ala7). As seen in Fig. 3, a single ion mobility peak was observed for both [kemptide+2H]2+ and [(Val6, Ala7)-kemptide+2H]2+. The calculated CCS values from the individual heptapeptide target analyte ions were 314 Å2 for [kemptide+2H]2+ and 311 for [(Val6, Ala7)-kemptide+2H]2+ as reported in Table 1. These CCS values correspond to a difference of 0.98% and partial resolution in the mobility domain with the 3S-IS as shown in Fig. 3b. However, for the 2S-IS, the doubly protonated ions ([kemptide+2H]2+, [(Val6, Ala7)-kemptide+2H]2+) of the heptapeptide mixture were overlapped and hence remained unresolved in the mobility domain as demonstrated in Fig. 3a. We note that in comparison to the work done by Wu et al.,[22] the calculated CCS values for the doubly protonated ions showed a small difference of about 0.5% and hence no separation was achieved for the doubly charged ions ([kemptide+2H]2+, ([Val6, Ala7)-kemptide+2H]2+). For reference, the resolving powers for this mixture using the 3S-IS were 124 for [kemptide+H]+ and 117, 102 for [(Val6, Ala7)-kemptide+H]+ (corresponding to the first and last peak at m/z 772 in Fig. 3, starting from the left). For the 2S-IS, the resolving power for [kemptide+2H]2+ was 100 and 90, 60 for [(Val6, Ala7)-kemptide+H]+ (corresponding to the first and last peak at m/z 772 in Fig. 3, starting from the left). Whereas, the resolving powers for the individual species were 106 for [kemptide+H]+ and 98, 95 for [(Val6, Ala7)-kemptide+H]+. Overall, an increase in ion counts and slight increase in resolving powers was achieved with the 3S-IS in comparison to the 2S-IS for the heptapeptide ion mixture. Although baseline resolution of the doubly protonated ions of the heptapeptide analyte mixture was not achieved using the 3S-IS, clear distinction of the two heptapeptide analyte ions is observed in comparison to the 2S-IS, where the doubly charged ions of the heptapeptide mixture were completely overlapped. Raw signal traces for m/z 386 from the FT-IMMS experiments are shown in Fig. 4 for kemptide/ (Val6, Ala7)-kemptide ion mixture using the 3S-IS and 2S-IS. From Fig. 4, a continuous increase in ion signal is observed for the 3S-IS throughout the duration of the experiment. Whereas, in the case of the 2S-IS, the ion intensity remains somewhat constant throughout the course of the experiment. This observation trends with our prior publication, where a comparison plot of the raw signal traces was made for the TXA, T5 ion using the 3S-IS and 2S-IS.[21] Fig. 4 and the data reported in Table 2, additionally highlight the increase in ion counts gained for the target analyte ion mixture using the 3S-IS in comparison to the 2S-IS.

Fig. 3
figure 3

Mass selected mobility spectra of two isomeric methyl substituted heptapeptides obtained using the FT-IMMS experiment with a frequency sweep from 5 to 10,005 Hz for 8 min using the (a) two-state ion shutter (2S-IS) for the ion mixture, (b) tri-state ion shutter (3S-IS) for the ion mixture. (c) The two hepta-peptides were individually analyzed using the 3S-IS

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Fig. 4
figure 4

Raw signal traces for m/z 386 from the FT-IMMS experiments are shown for kemptide/(Val6,Ala7)-kemptide mixture using the 3S-IS and 2S-IS. The raw signal traces for the ion populations using 3S-IS clearly exhibit two different ion populations. A polynomial xoffset fit is fitted to each signal trace just for the purpose of demonstrating the rise in ion signal

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Conclusion

Using an atmospheric, dual gate drift tube ion mobility coupled to a linear ion trap mass spectrometer using linear frequency modulation and the Fourier transform to recover gas-phase ion mobilities, we compare the separating capacity of system using the 3S-IS and 2S-IS principles for three pairs of peptide mixtures. From the results of the mobility separations, we illustrate the ability of multiplexing systems coupled with 3S-IS to enhance mobility separations and ion current. For each set of the isomeric peptides, both single and double protonated ions were observed in the mobility domain. For the three pairs peptide mixtures analyzed, the ion counts recorded for the singly and doubly protonated ions were higher with the 3S-IS compared to the 2S-IS. Similarly, the resolving powers for the target analyte ions were slightly higher (21%) with the 3S-IS in comparison to the 2S-IS. Compared to previous efforts of separating the same sets of isomeric peptides using drift tube IMMS with a Bradbury-Nelson ion shutter, the present work focuses on the benefits of using tri-state ion shutter (3S-IS) in FT-based IMMS experiments for complex analytical separation such as isomeric species. Separations achieved for isomeric peptides with the same m/z and charge state were due to small differences in their gas-phase structural conformation measurable as ion-neutral collision cross section (CCS). Under the outlined conditions, the resolving power of the instrument with the 3S-IS configuration used in this study allows separation of isomeric compounds with less than 1% differences in their CCS values.