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. Author manuscript; available in PMC: 2025 Aug 30.
Published in final edited form as: J Am Soc Mass Spectrom. 2025 Aug 21;36(9):1988–1994. doi: 10.1021/jasms.5c00246

Collision-Induced Unfolding of High-m/z Native-like Protein Ions within a Trapped Ion Mobility Spectrometer

Olakunle O Akinola 1, Nicholas B Borotto 1,*
PMCID: PMC12396232  NIHMSID: NIHMS2106692  PMID: 40839858

Abstract

Native mass spectrometry (nMS) is a powerful tool for the rapid characterization of protein ions and protein-ligand complexes. By coupling nMS with ion mobility spectrometry (IMS), and collisional activation, insights into protein conformation and stability can be rapidly obtained. Originally incapable of this workflow, recent work enabled this collision-induced unfolding (CIU) process on commercially available Bruker timsTOF instruments. This early work, however, faced challenges in transmitting larger proteins and only sought to unfold small proteins up to 29 kDa. In this study, we continue the development of this technique and optimized instrument settings to enable the transmission of proteins up to 8,000 Th. The technique also demonstrates the capability to sufficiently energize ions to unfold native-like dimers of superoxide dismutase and β-lactoglobulin, and the 45 kDa monomeric ovalbumin. When this TIMS activation technique is applied to large protein ions, however, limited unfolding was observed for bovine serum albumin and no unfolding was observed for immunoglobulin G likely reflecting the limit of activation for this workflow.

Graphical Abstract

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Introduction

Native mass spectrometry (nMS) and ion mobility spectrometry (IMS) have emerged as powerful tools for the high-throughput analysis of protein ions.1–5 nMS aims to retain and transfer the native structure of proteins into the gas phase, which is accomplished by employing nondenaturing solvents and by minimizing transfer voltages and the acceleration of ions within the mass spectrometer. The rotationally averaged collision cross section (CCS) of these protein ions can then be measured with IMS. This measurement provides insights into the initial solution-phase conformation of the protein.6–10

Collisionally activating these protein ions prior to the IMS measurement can dissociate the protein’s noncovalent bonds, induce protein unfolding, and lead to an increase in the measured CCS.11–13 The energy required to instigate this collision-induced unfolding (CIU) provides valuable information about the stability of the folded protein structure. These energies are influenced by factors such as post-translational modifications, ligand binding, and protein conformation.14–17

Trapped ion mobility spectrometry (TIMS) is one of the more recently developed IMS implementations and has found widespread adoption due to its high mobility resolution and relatively small physical footprint. These instruments, however, were not designed to activate ions prior to the IMS measurement and could not be used for the CIU workflow. This was initially overcome by Fernandez-Lima’s group by orthogonally accelerating ions as they exited the capillary towards the entrance funnel.18 This early work and work by others demonstrated that CIU could be instigated, but the maximum energies available to this technique were limited.18–25

Later studies by our group demonstrated that manipulation of the tunnel-in pressure and the voltage used to transfer ions between the accumulation and analytical trapping fields enabled precise titration of ion energies and fragmentation of protein and peptide ions.26–30 This activation occurred prior to mobility separation and all generated product ions were mobility separated. When this activation technique was applied to native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase, it promoted robust unfolding of each protein ion.31,32 in this work, we were able to measure native CCS values and generate CIU fingerprints that were consistent with prior works performed on other IMS instrumentation.31 The results from the TIMS activation technique were promising, but we were unable to transmit ions above ~4,000 Th and the ability of this activation technique to unfold protein ions greater than 29 kDa was still unexplored. Recent work has demonstrated that the 4,000 Th limit can be surpassed with an lower frequency RF generator, but this capability is not available to commercial instrumentation.33

Here, we optimize several instrumental parameters to improve transmission of large native-like protein ions on a commercial timsTOF. Intriguingly, the setting most critical to efficient transmision through the TIMS device was accumulation ramp height. With these optimized settings, we observe the transmission of protein ions greater than 8,000 Th. We also subjected protein ions and protein-protein complexes ranging in size from 31 to 150 kDa to collisional activation. When activated, all protein and protein complex ions up to 45 kDa underwent robust unfolding. When bovine serum albumin (BSA) and immunoglobulin G (IgG) were collisionally activated, however, BSA only undergoes partial unfolding and IgG undergoes no unfolding even at the highest energies.

Experimental Section

Materials and Reagents

Albumin from bovine serum (BSA), β-lactoglobulin from bovine milk, and superoxide dismutase from bovine erythrocytes, were all obtained from Sigma-Aldrich (St. Louis, MO). Recombinant humanized IgG1κ, expressed in murine suspension, was purchased from Agilent (Cedar Creek, TX). Albumin from egg white (ovalbumin, OVA), ammonium acetate, and mass spectrometry-grade Optima water were all acquired from Fisher Scientific (Waltham, MA). Each protein, with the exception of βLG, was diluted to a final concentration of 6 μM in 100 mM ammonium acetate. βLG was prepared in 200 mM ammonium acetate.

Ion Mobility Spectrometry and Mass Spectrometry

All experiments were performed on a Bruker (Billerica, MA) timsTOF. The capillary voltage, end plate offset, nebulizing gas pressure, dry gas flow rate, and dry temperature were set to 4000 V, 400 V, 1.5 bar, 4 L/min, and 150°C, respectively. The mobility scan time was set to 100 ms for SOD and βLG, to 70 ms for OVA, and to 60 ms for BSA and IgG. CIU fingerprints were obtained by increasing the Δ6 DC voltage in 5 V increments from 30 to 150 V, while the other TIMS transfer DC voltages were set as follows: Δ1 = −10 V, Δ2 = −70 V, Δ3 = 20 V, Δ4 = 20 V, and Δ5 = 0 V. The ion mobility accumulation time was set to 40 ms for SOD and βLG, and the ICC values were set to 2.5 million ions (mio), 3.0 mio, and 5.0 mio for BSA, OVA, and IgG, respectively. The RF amplitude was maintained at 350 Vpp. The mobility scan range for IgG spanned from 1/K0 = 0.5 to 4.0 V·s/cm2, while for OVA, SOD, βLG dimer, and BSA, it ranged from 1/K0 = 0.50 to 2.50 V·s/cm2. All experiments were conducted at 1.5 mbar tunnel-in pressure, with the instrument calibrated using Agilent tune mix for both mass and mobility prior to each experiment.

Data analysis

Ion mobility spectra were manually exported and analyzed using CIUsuite 2.34 CIU fingerprints were constructed by averaging three technical replicates collected over three days. All replicates were simultaneously loaded into the software and compared pairwise. The average root-meansquare deviation (RMSD %) for each comparison were calculated and reported. To determine the collisional cross-section (CCS) of native-like ions, mobility values were collected in triplicate at Δ6 = 30 V and converted to CCS using the Mason-Schamp equation. These values were used to generate plots, where the CCS peaks were identified and fitted using the multiple peak fitting tool in OriginPro 2021. The average centroid for each fitted peak was then reported. All reported CCS values in this study should be considered TIMSCCSN2 based on the recent recommended nomenclature.35

Results and Discussion

The manner by which the mobility separation is achieved in TIMS has been the subject of multiple reviews and we suggest reviewing these excellent works for a detailed explanation.36–39 While the separation process utilized here does not deviate from these prior works, the analysis of native-like protein ions requires slight modifications to this process. In particular, protein analytes are subjected to multiple acceleration voltages prior to the IMS measurement and care must be taken to ensure the native-like conformation persists through each step; as the final measured conformation is dependent solely on the most activating step. The variables that lead to activation and unfolding have been extensively studied in works by Morsa et. al., Bleiholder et. al., Borotto et. al. and later by Panczyk et. al.25,31,40,41 These studies found that the transfer voltages Δ3, Δ4, and Δ6 each possessed the ability to stimulate sufficiently energetic collisions to activate ions. Thus, to minimize this activation and ensure that native CCS values are measured, these transfer voltages were initially set to 20, 20, and 30 V, respectively for all following experiments (Scheme 1). In our prior work, we demonstrated that the systematic increase of the Δ6 voltage promoted the stepwise unfolding of protein ions up to carbonic anhydrase (29.5 kDa). This region of the instrument was not designed to activate ions, however, and we anticipate that the energies achievable by this workflow are limited and as the mass and degrees of freedom of our analytes increases, this technique will no longer be able to achieve sufficient energies to unfold these ions. To discover the mass limit of this activation technique, we analyzed a series of sequentially larger protein ions, starting with superoxide dismutase (SOD) a 15.7 kDa protein that readily forms dimers. When SOD is directly infused into the mass spectrometer, the 5, 6, and 7+ charge states of the protein monomer and 11+ charge state of the dimeric species are observed (Figure 1A and Figure S1). The CCS values for the 5+ and 6+ charge states are measured as 1307 ± 4 Å2 and 1470 ± 30 Å2, respectively, while the 11+ dimer had a CCS of 2876 ± 18 Å2. While the CCS of the dimer is approximately 3% larger than the combined CCS of the 5 and 6+ ions, the measured values for the 6+ and 11+ ions (no previous CCS value has been reported for the 5+ monomer) are within 5.4% and 4.9%, respectively, of the previously measured values (Table S1).42 This indicates that our current settings are capable of retaining the native-like conformation of this protein and protein complex. To examine if this technique is capable of unfolding this 31 kDa protein complex, we systematically increased the Δ6 voltage and indeed observed the unfolding dimer (Figure 1B). For the 11+ ion, two transitions and three distinct features were populated as the Δ6 voltage was increased from 30 to 150 V. These three observed features are consistent with previous data acquired on a Waters Synapt.43 As discussed in our prior work, since there is no isolation step prior to the IMS measurement, all generated ions are activated simultaneously.31 Thus, while our focus was the protein dimer, the monomeric ions were also extensively activated and unfolded as the Δ6 voltage increased (Figure S2). This enables facile comparison of all oligomeric species present as all will be assessed in a single experiment.

Scheme 1. (A) Schematic of TIMS device with the location of salient DC transfer voltages indicated. (B) Illustrates the electric field gradients (EFG) that ions are subjected to during the TIMS analysis. (B. Top) analyte ions are initially trapped on the accumulation ramp. (B. Middle) the trapped ions are released from the accumulation region transferred to the analytical EFG. (B. Bottom) Ions are initially trapped and then separated on the analytical EFG and systematically released through stepwise reduction of the trapping field. Protein model PDB accession codes: 6EYY and 6LKF.

Scheme 1.

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Figure 1: A) Mass spectrum of superoxide dismutase with the 5+, 6+, and 7+ charge states for the monomer and the 11+ charge state for the dimer observed. B) CIU fingerprint of the 11+ dimer of SOD.

Figure 1:

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To examine a larger protein complex, β-lactoglobulin (βLG) was prepared in 200 mM ammonium acetate and when this protein solution was directly infused into the mass spectrometer, monomeric cations of the two most prevalent isoforms44,45 with between six and nine protons added, and the 12+ and 13+ charge states of the protein dimer are observed (Figures 2A and S3). When the 13+ dimer is assessed with TIMS, we observe a single mobility feature at 3408 ± 6 Å2, which is within 4.1% of a CCS value acquired on a drift tube (Table S1).46 This dimer has a mass of 36.8 kDa and represents a significant increase in mass from the prior assessed proteins. Despite the increase in size, when Δ6 is increased from 30 to 150 V, the 13+ charge state of the dimer unfolds in a single step to a more extended conformation centered at 4835 Å2 (Figure 2B). When analyzing oligomeric protein ions, monomeric and higher-order oligomers can overlap at the same m/z value and when the 6+ monomer is examined closely it becomes apparent that a 12+ dimer is also present at this m/z (Figure S3). When assessed with TIMS, we observe a mobility spectrum that while not fully resolved does indeed correspond to partially overlapping dimer and monomer features (Figure S4). When this mixture of monomeric and dimeric ions is collisionally activated, both the monomer and dimer simultaneously progress from a folded to an unfolded conformation producing a convoluted CIU fingerprint where it is difficult to assign the observed features to a specific species (Figure S5). Typically, the shared m/z values of these oligomers lead to these ions being avoided when performing native mass spectrometry measurements; but, while not ideal, in situations where there are overlapping oligomers, this data can be deconvoluted by extracting the mobility spectrum for an isotope that can be only attributed to the higher charged dimer. To demonstrate this, we extracted the mobility spectra for the most abundant isotope with a 0.03 Th width that could only correspond to the dimer at each activation voltage and plotted a CIU fingerprint. We performed the same action for an isotope that corresponded to both the monomer and dimer but should predominately reflect the behavior of the monomer (Figure S6). When these two fingerprints are compared the differences between the unfolding behavior of the dimer and monomer are apparent (Figure S6). While the exclusion of the other isotopes and a majority of a protein’s ion abundance causes the signal-to-noise ratio of these CIU fingerprints to suffer, this is an effective method for extricating a higher-order oligomer’s behavior from an overlapping lower-order oligomer. This technique is also useful for acquiring accurate CCS values for these overlapping features and we measure the CCS of the 6+ and 12+ ions as 1610 ± 4 Å2 and 3320 ± 80 Å2, respectively, which are within −1.6% and 5.1% of the reported literature value (Table S1).46,47 Interestingly, the dimer again appears to be approximately 3% larger than the CCS of two monomers combined, suggesting there may be a systematic over estimation of dimer CCS values.

Figure 2: A) Mass spectrum of native-like βLG. The 6+ through 9+ reflect the monomeric protein and the 13+ charge state reflects the dimer. The A and B isoforms are indicated in black and red. B) CIU fingerprint of the 13+ charge state of the βLG dimer.

Figure 2:

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To further explore the mass limit of this activation technique, we next applied it to the monomeric ovalbumin (OVA), a 45 kDa glycoprotein. When ionized from a non-denaturing solution, we observe a collection of charge states ranging from the 12+ to the 15+ (Figure 3A). This protein is known to be post-translationally modified48–50 and several proteoforms are observed (Figure S7). Each of the observed four charge states have a single mobility feature measured at 3424 ± 14 Å2, 3474 ± 13 Å2, 3488 ± 15 Å2, and 3540 ± 30 Å2 for the 12, 13, 14, and 15+, respectively (Table S1). Previous reports measured the CCS of ovalbumin using a drift tube with helium and as expected for results acquired with helium,51 the reported CCS values are significantly lower with CCS values of 3010 Å2, 3040 Å2, and 3050 Å2 being reported for the 12+, 13+, and 14+ charge states, respectively.52 When compared to the CCS of the 12+ ion reported by Vallejo et al. our results are within 1.9% of this previously reported value of 3360 Å2.53 As in our previous protein ions, when this 45 kDa protein is subjected elevated voltages, each charge state is collisionally activated and we observe conformational isomerization and an increase in the measured CCS values of all ions. When this energy is systematically increased, each charge state gradually unfolds, enabling the generation of a CIU fingerprint for each charge state (Figure 3B, C and Figure S8). These results indicate that the timsTOF is indeed capable of instigating the unfolding of protein and protein complexes up to 45 kDa.

Figure 3. A) Mass spectrum of ovalbumin ionized out of native conditions. CIU fingerprints of the B) 12+ and the C) 13+ charge states of ovalbumin.

Figure 3.

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Larger native-like protein ions often have mass-to-charge ratios greater than 4000 Th and in our previous work, the instrument was incapable of transmitting and measuring proteins with mass-to-charge ratios of this magnitude.31 Thus, to assess these larger protein ions, this limitation must be overcome. When the TIMS is configured to transmit ions and not perform an IMS measurement, this instrument is able to transmit these more massive protein ions, suggesting that they are lost during the ion mobility measurement (data not shown). We initially believed that this was a fundamental limitation of the instrument, and begin to investigate lower frequency RF generators as recently demonstrated by the Wysocki group.33 But, as the instrument was frequently alternated between high- and low-mass configurations, it was serendipitously discovered that high-mass ions could be transmitted with the commercial RF generator. Investigation into the critical setting discovered that the maximum accumulation ramp height, was the critical variable. As the name suggests, this controls the maximum voltage for the accumulation electric field gradient (EFG) and is typically coupled to the height of the analytical EFG (Scheme 1). When BSA is directly infused and this value is set to 4.5 V·s/cm2 no signal is detected, but, when this voltage is decoupled from the analytical EFG and systematically decreased, we observe a corresponding increase in the ion signal detected (Figure 4). When this max accumulation height is set to a value of 2.5 V·s/cm2 and BSA is directly infused, the 15+, 16+, 17+ and 18+ charge states are the major species generated and detected (Figure 5A). The CCS values measured for these ions were 4450 ± 140 Å2, 4583 ± 13 Å2, 4660 ± 15 Å2, and 4746 ± 12 Å2 for the 15+, 16+, 17+ and 18+, respectively. These measured CCS values for the 15+ and 16+ ions were within −0.7% and 2.7%, respectively, of prior TIMS measurements,54 and the 15+, 16+, and 17+ states are on average within 1% of values measured previously on a drift tube (Table S1).46 While transmission of these large ions is undoubtedly a positive outcome, we are still unsure how to explain this behavior. We initially hypothesized that reduction of this voltage results in a shallower slope for this trapping field which could better distribute ions in space and decrease space charge, but other methods of reducing ion count do not substantially improve ion transmission, so this appears to be incorrect. We will continue to pursue the basis of this behavior in future work.

Figure 4: Ion abundance the 15+, 16+, and 17+ charge states of BSA at the indicated accumulation ramp height.

Figure 4:

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Figure 5: A) Mass spectra and B) CIU fingerprint of the 17+ charge state of native-like BSA. (C) Mass spectrum of IgG ionized from native conditions. D) CIU fingerprint of the 22+ charge state of IgG demonstrating that the instrument is incapable of reaching energies sufficient to unfold these ions.

Figure 5:

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With transmission of native-like ions of BSA achieved, we next sought to perform CIU. Upon increasing the Δ6 voltage, ion activation and conformational isomerization was indeed observed. The 16+ and 17+ ions exhibited a single transition state and unfolded into a more extended conformation centered at approximately 5500 Å2 (Figure 5B and Figure S9). When this result is compared to previous studies, it appears that this technique cannot access sufficient energies to completely unfold this protein. These prior works indicate that BSA undergoes multiple unfolding transitions while the TIMS is only capable of reaching energies sufficient to access the first unfolding transition.16,55 As discussed earlier, these larger proteins are more difficult to unfold due to the increased number of noncovalent bonds and degrees-of freedom and it appears that we are approaching the limit of this activation technique.56–59

While this technique is unlikely to access sufficient collisional energy to promote the unfolding of a 150 kDa IgG, we pursued this protein next to explore if this instrument is capable of performing native CCS measurements of antibodies. And indeed, when the accumulation ramp height is maintained at its reduced value and an IgG is directly infused into the instrument, we detect multiple charge states ranging from 19+ to 29+ (Figure 5C). When these IgG ions are assessed with IMS, the CCS values are measured as 6100 ± 80 Å2, 6300 ± 50 Å2, 6510 ± 40 Å2, 6690 ± 30 Å2, and 6864 ± 17 Å2 for the 22+, 23+, 24+, 25+, and 26+ ions, and these ions are within −15%, −12%, −10%, and −9% of the values collected on a prototype TIMS instrument (Table S1).54 These CCS measurements show greater deviation from prior results, and we will investigate the source of this error in future work. Lastly, to examine if the timsTOF is capable of instigating unfolding of an IgG, we steadily increased the Δ6 voltage and observed no change in the measured CCS value (Figure 5D). This suggests that the collisional energy available on the current instrument is insufficient to induce unfolding of high-mass ions like IgG.

Conclusions

In this work, we demonstrate that the Bruker timsTOF is capable of unfolding protein ions and protein dimers up to 45 kDa. We also demonstrated that by extracting the mobility spectrum of a single isotope, CIU fingerprints of overlapping oligomeric species could be extricated enabling the assessment of both the lower- and higher-order oligomers independently. Next, we uncovered that the accumulation ramp height is a critical setting in effectively transmitting high-m/z ions through the TIMS device. By decoupling the height of the accumulation ramp from the analytical ramp and lowering the value of the accumulation ramp from 4.5 to 2.5 V·s/cm2, native ions of BSA and an IgG up to 8,000 Th, could be transmitted and measured. Lastly, we examined if the TIMS device can unfold these large protein ions and found that the TIMS possessed sufficient energy to partially unfold BSA but was incapable of unfolding IgG. This likely represents a fundamental limitation of these early dual-TIMS based instruments.

Supplementary Material

Supporting information

An additional table, plot, and references comparing measured and literature CCS values can be found in the supplemental information. These items can be found online free of charge at.

ACKNOWLEDGMENT

Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R35GM151104. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data Availability Statement

Raw mass and mobility spectra can be found under the accession code MSV000098552 in the MassIVE data repository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information
NIHMS2106692-supplement-Supporting_information.pdf (960.4KB, pdf)

Data Availability Statement

Raw mass and mobility spectra can be found under the accession code MSV000098552 in the MassIVE data repository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp).

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