Direct Observation of Metastable Fragment Ions in Ultraviolet Photodissociation of Ubiquitin
More
Advanced Search
  • Free to Read
  • Editors Choice
Article

Direct Observation of Metastable Fragment Ions in Ultraviolet Photodissociation of Ubiquitin
Click to copy article linkArticle link copied!

Open PDFSupporting Information (1)

Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2026, 37, 4, 910–918
Click to copy citationCitation copied!
https://doi.org/10.1021/jasms.5c00432
Published March 11, 2026

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved. This publication is licensed under these Terms of Use.

Request reuse permissions

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Ultraviolet photodissociation (UVPD) of proteins is known to exhibit conformation-dependent fragmentation patterns, but direct structural evidence linking precursor protein and fragment ions has been limited. Here, we apply tandem trapped-ion mobility spectrometry/tandem-mass spectrometry to compare collision cross sections of UVPD fragment ions generated from distinct conformers of ubiquitin. Under the high-pressure (∼4 mbar) and low-photon density (∼10 μJ laser pulse energies) conditions employed here, UVPD produces predominantly [b + 2] and [y – 2] ions at proline residues, consistent with direct bond cleavage from the electronically excited state. Our data show that these ions can retain a clear structural relationship to the precursor conformation: UVPD of compact, native-like ubiquitin yields fragments with collision cross sections ∼20% smaller than the corresponding ions produced from extended precursors or by collision-induced dissociation. Further, these compact UVPD fragments are kinetically trapped in metastable conformations, with substantial barriers preventing relaxation toward energetically favored gas-phase structures. We attribute this behavior to limited vibrational energy deposition per absorbed 213 nm photon combined with rapid collisional cooling, which suppress cumulative thermal activation and disfavor statistical fragmentation pathways, leaving direct excited-state dissociation as the dominant observable process. Together with prior UVPD studies on holo-myoglobin, our results suggest that UVPD fragments can retain aspects of their precursor tertiary structure.

This publication is licensed for personal use by The American Chemical Society.

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.

Subjects

what are subjects

Special Issue

Published as part of Journal of the American Society for Mass Spectrometryspecial issue “Fenn: Photoactivation and Ion Activation”.

Introduction

Click to copy section linkSection link copied!
Mass spectrometry (MS) is now widely applied to probe protein structures. (1,2) A key advantage of MS is that it can detect transient and sparsely populated species. (3−5) Transient species often underlie protein function and regulation, (6−9) but remain difficult to resolve with established techniques. (7,9)
The cornerstone of MS–based methods to protein structure is tandem-mass spectrometry. Its success rests on a diverse suite of ion activation methods, from slow-heating collision-induced dissociation (CID) (10) to faster activation strategies such as electron-capture/transfer dissociation (ECD/ETD), (11) surface-induced dissociation (SID), (12) or UV photodissociation (UVPD). (1)
UVPD has emerged as a powerful approach for top-down analysis of proteins and their complexes. (13−15) In a typical UVPD experiment, (16,17) UV photons are absorbed by protein amide bonds, amide bonds dissociate, and the resulting fragment ions are identified by MS. A major advantage of top-down protein analysis with UVPD is its ability to generate fragments from internal regions of the protein, often yielding exceptionally high sequence coverages. (13)
Beyond primary structure, UVPD can also report on protein tertiary and quaternary structure when performed under native MS conditions. Differences in fragment-ion patterns between two states of the same protein─for example, ligand-bound versus ligand-free─can reveal changes in conformation or ligand-binding. (15,18,19) This capability makes UVPD a versatile tool for probing both sequence and higher-order structural features in biomolecular systems.
A striking feature of UVPD under native MS conditions is that fragment ion identities and abundances can correlate with structural features of the solution-phase protein, such as X-ray B-factors. (15) These correlations indicate that protein backbones may not reorganize significantly during UVPD and instead report selectively on flexible versus rigid regions of the protein structure. Equally intriguing are reports that the UVPD fragment pattern depend on the conformation adopted by the precursor ions in the solvent-free environment. For example, Brodbelt and co-workers combined ion mobility spectrometry with UVPD tandem mass spectrometry and showed that the specific precursor conformation has a pronounced impact on which fragments are observed and in what abundances. (20,21) Moreover, UVPD spectra from the Brodbelt (18) and Barran (22) laboratories indicate that certain noncovalent tertiary interactions present in the native state of holo-myoglobin can be preserved in UVPD fragments.
While these prior studies have demonstrated that UVPD fragment yields and cleavage patterns can depend on precursor conformation, fragment ions are often implicitly assumed to relax rapidly to energetically favored conformations in the gas phase following dissociation. Whether fragment ions can instead remain kinetically trapped in conformations that retain structural memory of their precursor remains largely unexplored. Establishing the existence of such metastable fragment ions would therefore have important implications for structural mass spectrometry, as it would suggest that fragment ion collision cross sections may yield information about the structure of the intact protein.
Characterizing the structures of fragment ions requires an experimental platform that can (i) resolve and characterize precursor protein conformations, (ii) perform UVPD on a selected precursor conformation, and (iii) identify and structurally characterize the resulting UVPD fragment ions. Our tandem-trapped ion mobility spectrometry/tandem-mass spectrometry platform (23,24) (Tandem-TIMS, Figure 1) provides exactly these capabilities by mobility-analyzing protein conformations in TIMS-1, mobility-selecting a specific conformation, subjecting the selected ions to UVPD, and then mobility-analyzing the resulting fragment ions. Here, we apply Tandem-TIMS to ask whether UVPD-generated fragment ions can retain a discernible structural relationship to their precursor. To address this question, we performed UVPD of selected conformers of the prototype protein ubiquitin and characterized the structures of the produced fragment ions. The 76-residue α/β protein ubiquitin (8.6 kDa) is an ideal model system: its tertiary structure comprises an α-helix and a β-sheet with five antiparallel β-strands, (25,26) it is stable in a broad range of solution conditions, (27) and it has been extensively characterized in solvent-present and solvent-absent environments.

Figure 1

Figure 1. (A) NMR solution structure of the model protein ubiquitin used in this study. (B) Schematic of the tandem-trapped ion mobility spectrometer/tandem-mass spectrometer coupling two trapped ion mobility spectrometers (TIMS-1, blue; TIMS-2, red), an ion trap for ion storage, and a UV laser for photodissociation of the stored ions (green). This platform allows characterizing the precursor protein conformations, performing UVPD of a selected precursor conformation, and subsequently characterizing the structures of the produced fragment ions.

High Resolution Image
Download MS PowerPoint Slide

Materials and Methods

Click to copy section linkSection link copied!

Materials and Sample Preparation

Ubiquitin from bovine erythrocytes (≥98%) and water (LC/MS grade) were obtained from Sigma-Aldrich (St. Louis, MO). Acetic acid (glacial) was obtained from Fisher Scientific (Pittsburgh, PA). High-concentration ESI tuning mix was obtained from Agilent (Santa Clara, CA). An aqueous ubiquitin solution was prepared at a concentration of 10 μM with 1v% acetic acid for the measurements. ESI tuning mix was used as obtained for mass and ion mobility calibration. (28,29)

Tandem-TIMS Measurements

Experiments were performed on a previously described orthogonal Tandem-TIMS (Figure 1B) (23,30) constructed from adding a separate trapped ion mobility spectrometry (TIMS) (31) device to a timsTOF Pro (Bruker Daltonics, Billerica, MA) and coupling with a Nd:YAG laser producing 213 nm photons (NL204, EKSPLA, Vilnius, Lithuania). Protein ions are generated using native electrospray ionization and are gently transferred through the instrument. (32−34) Tandem-TIMS (23,24) first separates distinct protein conformations and measures their collision cross sections in TIMS-1. Electrodes downstream of TIMS-1 select the desired protein conformation after elution from TIMS-1, (24) followed by storing the selected protein ions in the ion trap and irradiating them with UV photons. If the stored protein ions absorb UV photons and dissociate into fragments, the collision cross sections of the fragments are measured in TIMS-2. (23,30) Alternatively, the selected protein conformation can be collisionally dissociated (CID). (23,24,30,33,34) The resulting fragment ions can optionally be collisionally activated by applying strong electric potentials between the T2 and T3 electrodes in TIMS-2 (Figure 1B). (35) Collision cross sections of the fragment ions are subsequently measured in TIMS-2. UV photodissociation was achieved by 213 nm photons produced from the fifth harmonic of a Nd:YAG laser operating at 1 kHz. Mobility-selected ubiquitin precursor ions were confined in the ion trap for 50 to 500 ms and exposed to approximately 50 to 500 laser pulses. For collision-induced dissociation, the selected, intact ubiquitin precursor ions were collisionally dissociated by placing a voltage difference of 180 V between apertures L3 and V1 (Figure 1B). (30) Collision cross sections in TIMS are determined via a calibration procedure as described. (36−28) Mobility calibrations were carried out using phosphazene ions contained in ESI tuning mix. (37) Furthermore, all UVPD spectra were recorded without lasing in the first ∼30 s– 60 s to allow the molecular ubiquitin ions of charge states 8+ and 7+ to be used as internal calibrants. All theoretical isotope patterns were calculated using the EnviPat algorithm. (38)

Computational Details

Langevin dynamics simulations were carried out with GROMACS 4.5.742 in conjunction with the OPLS/AA force field (39,40) to assess intramolecular vibrational energy redistribution and collisional energy transfer between a polypeptide and the N2 buffer gas at an experimental pressure of ≈4 mbar. Nitrogen parameters were taken from a prior report. (41) The peptide and N2 molecules were placed in a cubic box (120 × 120 × 120 nm3) with periodic boundary conditions, and the N2 number density was adjusted to reproduce a pressure of ≈4 mbar. A time step of 0.75 fs was used to treat all vibrational modes during energy dissipation explicitly. The polypeptide was not coupled to an external thermostat, so its temperature evolved freely during the simulation. In contrast, all N2 molecules were weakly coupled to a 300 K heat bath via a large Langevin friction coefficient, maintaining an isothermal N2 environment while allowing collisional energy exchange with the peptide. Under these conditions, vibrational cooling or heating of the peptide occurred exclusively through peptide–N2 collisions. Cutoffs of 20 nm were applied to electrostatic and van der Waals interactions. Positions, velocities, and energies were saved at intervals sufficient to monitor the temperature relaxation of the peptide. Each simulation was propagated for up to three microseconds to ensure that vibrational relaxation was fully converged. No center-of-mass motion removal was applied to avoid biasing energy transfer between the peptide and N2 particles. This simulation scheme isolates collisional energy transfer as the sole vibrational energy relaxation mechanism for the peptide, to allow direct assessment of collisional-vibrational energy transfer between a vibrationally hot polypeptide and the N2 buffer gas under our experimental conditions. (39−41)

Results and Discussion

Click to copy section linkSection link copied!

Direct Cleavage from the Photoexcited State Predominates UVPD in Tandem-TIMS

UVPD measurements were carried out on our tandem-trapped ion mobility spectrometer (Tandem-TIMS, Figure 1B) coupled with an ion trap, a Nd:YAG laser producing 213 nm photons for UV photodissociation of the trapped protein ions, and a tandem-mass spectrometer. (23) As expected from prior reports, charge states 7+ and 8+ predominate the mass spectrum of ubiquitin (Figure 2A). A compact conformation predominates charge state 7+, whereas charge state 8+ shows both a compact and an extended conformation. The compact features correspond to native-like conformations, (34,42−44) whereas the extended features correspond to gas-phase structures formed from the unfolding of the compact conformation in the absence of solvent, as demonstrated in prior mobility-selective activation experiments. (34) The presence of two strongly different ubiquitin conformations renders charges state 8+ ideally suited to probe if UVPD preserves a structural relationship between precursor and fragments.

Figure 2

Figure 2. Conformationally selective UVPD of ubiquitin in Tandem-TIMS. (A-i) Native ion mobility/mass spectrometry of ubiquitin showing charge states 7+ and 8+. (ii) Charge state 7+ exhibits mainly a native-like conformer (blue), whereas (iii) charge state 8+ shows a dominant native-like conformation (1283 Å2, blue) and a minor gas-phase structure (1947 Å2, orange). (B-i) Conformationally selective top-down mass spectrometry of ubiquitin charge state 8+. (ii) CID produces typical a-, b-, and y-type fragments, whereas UVPD of the (iii) native-like and (iv) gas-phase ubiquitin conformations shows formation of [a + 2], [b + 2], and [y – 2] fragment ions.

High Resolution Image
Download MS PowerPoint Slide
We thus selected each of the conformations of charge state 8+ after elution from TIMS-1 (Figure 2B), (24) and then dissociated them either by UVPD or CID (as control). For CID, the selected ions were collisionally activated by voltages applied across electrodes L3 and V1 in the TIMS-1/TIMS-2 interface (Figure 1B). This produced an, bn, yn - type ions that are typically observed for CID of a polypeptide. (23,24,45,30)
By contrast, UVPD (Figure 2B) in Tandem-TIMS did not produce an, bn, yn-type fragment ions. Instead, UVPD produced primarily [b36+2]4+ and [y40-2]4+ ions from cleavage N-terminal to Pro37, and [y58-2]5+ ions from cleavage N-terminal to Pro19. This is consistent with prior literature, (46) as is the formation of [bn+2] and [yn-2] fragments for UVPD of proline-containing polypeptides. (16,17,47) Further, experimental (48) and theoretical (16) evidence indicate that these ion types are produced by direct-cleavage from the photoexcited electronic state on a nanosecond time scale.
We next probed whether the [bn+2] and [yn-2] fragment ions are exclusively produced when the ubiquitin precursor ions are UV irradiated while they are stored in the ion trap between TIMS-1 and TIMS-2. To this end, we confirmed that these ions are not produced in the absence of UV irradiation (Figures 3A, 3D) nor if the precursors are irradiated but not stored in the ion trap (Figure 3E). These results show that the [bn+2] and [yn-2] fragment ions are observed only when ion-trap storage is combined with UV irradiation; neither UV irradiation without trapping nor purely collisional activation under otherwise identical conditions generates these fragment ions.

Figure 3

Figure 3. y585+and [y58 – 2]5+ fragment ion isotope patterns under various measurement conditions. (A) CID in the TIMS-1/TIMS-2 interface produces y585+ ions exclusively. (B, C) UVPD of the compact and extended conformations produces [y58 – 2]5+ fragment ions but no y585+ ions. (D, E) The [y58 – 2]5+ fragment ions are not observed if the ions (D) are not irradiated by UV photons or (E) are irradiated without storage in the ion trap.

High Resolution Image
Download MS PowerPoint Slide
Taken together, the absence of traditional CID-type fragment ions in our UVPD spectra strongly indicate that dissociation from a vibrationally excited electronic ground state following UV photon absorption and internal conversion does not play a significant role in our measurements. Instead, our data strongly indicate that direct dissociation from an electronically excited state predominates in our UVPD measurements carried out at high-pressure (∼4 mbar) and low UV pulse energies (∼10 μJ). The principal UVPD fragments observed here originate from structurally distinct regions of ubiquitin. Mapping these fragments onto the crystallographic structure (Supporting Information, Figure S6) shows that they encompass both β-sheet and α-helical elements.

UVPD in Tandem-TIMS Preserves a Structural Relationship between Precursor and Fragment Ions

To characterize the structures of the produced fragment ions, we recorded their collision cross sections in TIMS-2 (Figure 4). The traditional bn and yn fragment ions generated by CID from the native-like (compact) and gas-phase (extended) ubiquitin conformations exhibit negligible differences in the number of spectral features, their collision cross sections, or their relative abundances (Figure 4A). This observation is in line with our prior reports of top-down protein analysis with Tandem-TIMS (24,45) and expected from the established mechanism of CID, (10) where successive low-energy collisions with buffer-gas neutrals gradually energize the protein. This gradual vibrational excitation promotes isomerization of a native-like protein conformation into gas-phase favored structures before and/or during amide bond cleavage. Hence, the observed fragment ions adopt energetically favored gas-phase structures irrespective of the precursor structure prior to dissociation.

Figure 4

Figure 4. (A) Ion mobility spectra of CID fragment ions produced from the compact (blue) and extended (orange) ubiquitin conformations agree closely. Hence, the produced fragment ions adopt the same gas-phase favored structures, irrespective of the precursor conformation. (B) Ion mobility spectra for the equivalent fragment ions produced by UVPD from the native-like conformation (blue) differ strongly from those produced from the extended, gas-phase conformation (orange). Here, a compact precursor produces fragments with compact conformations, whereas an extended conformation yields fragments with extended structures. This shows that UVPD can generate fragment ions that exist in a nonannealed, metastable conformation.

High Resolution Image
Download MS PowerPoint Slide
In contrast, our data show that [bn+2] and [yn-2] ions produced by UVPD from the native-like and gas-phase ubiquitin conformation─acquired under identical ion trapping and irradiation conditions─can differ significantly in their collision cross sections. Figure 4B shows the collision cross sections recorded for the principal fragment ions [b36+2]4+ and [y40-2]4+ from cleavage at Pro37 and [y58-2]5+ from cleavage at Pro37. These spectra show that UVPD of the compact, native-like ubiquitin precursor yields fragment ions that maintain a compact structure, with collision cross sections roughly 20% smaller than those of the fragments generated from the extended precursor.
These experimental observations show that the structures of these UVPD-generated fragments correlate with the precursor structure: UVPD of the compact precursor conformation produces fragment ions with compact conformations, whereas the extended precursor conformation gives rise to extended fragment ions.
Further analysis reveals that the compact UVPD fragments cannot be explained as gas-phase–annealed structures. As shown in Figure 4B, UVPD of the native-like precursor yields fragment ions with collision cross sections approximately 10–25% smaller than those produced from the gas-phase precursor under identical activation. If the fragments had annealed into their energetically favored gas-phase structures prior to detection, structural differences inherited from the precursor would be lost and fragments derived from either conformer would exhibit similar collision cross sections. Instead, the substantial collision cross section differences of the UVPD fragments indicate kinetically trapped, metastable conformations.

UVPD of Native-like Ubiquitin Produces Metastable Fragment Ions

The proposition that UVPD of the native-like ubiquitin conformation produces compact, metastable fragment ions that have not yet isomerized into energetically favored gas-phase conformations can be tested by examining their collision cross sections as a function of their vibrational activation: (49) If fragment ions are kinetically trapped in a metastable conformation, then they would require additional vibrational energy to overcome the activation barrier for structural rearrangement into their gas-phase favored structure. Thus, these initially compact ions would isomerize into an energetically favored gas-phase conformation upon vibrational activation. By contrast, if the compact conformation of the fragment ions is already in an energetically favored gas phase conformation, they will not undergo significant structural changes upon activation.
Thus, we probed whether collisional activation unfolds the compact UVPD fragment ions into their gas-phase favored structures. Figure 5 shows the spectra recorded for the [y40-2]4+ and [y58-2]5+ fragment ions as a function of collisional activation. The energetic activation was accomplished by placing a voltage of up to 140 V between electrodes T2 and T3 in TIMS-2 (Figure 1B). The spectra reveal that the compact [y40-2]4+ and [y58-2]5+ fragments produced from UVPD of the compact, native-like ubiquitin conformation increase in cross section by approximately 20% when collisionally activated by up to 140 V. The conformational transition from the initially compact conformation to the extended conformation takes place at voltages of about 60 V ([y40-2]4+) and 70 V ([y58-2]5+), respectively. At 140 V, the compact fragment ions become fully extended, yielding collision cross sections that agree with those produced by both CID and UVPD when applied to the extended gas-phase ubiquitin conformation.

Figure 5

Figure 5. Compact UVPD fragment ions produced from the native-like ubiquitin conformation are metastable. The compact (A) [y40 – 2]4+ and (B) [y58 – 2]5+ ions produced by UVPD from the native-like ubiquitin conformation require substantial activation to overcome an energy barrier and unfold into gas-phase favored conformations. The cross sections of the unfolded, gas-phase conformations are consistent with those of the extended fragment ions produced by UVPD from the extended gas-phase ubiquitin conformation and by CID.

High Resolution Image
Download MS PowerPoint Slide
Taken together, our data reveal that the initially compact fragment ions require significant energetic activation to overcome an activation barrier and reach gas-phase conformations. This underlines that the initially formed, compact UV photofragments are kinetically trapped in conformations that (i) differ significantly from the energetically favored gas phase conformations and (ii) are separated from the energetically favored gas phase conformations by a significant energy barrier.

Increased Photon Exposure Increases Fragment Yield but Does Not Change Fragment Conformation

To further examine whether UV photon absorption influences fragment ion structure, we systematically increased the UV irradiation time of the mobility-selected ions by increasing the trap storage time from 50 to 500 ms. These storage times correspond to UV irradiation of the stored ions by approximately 50 to 500 laser pulses at 1 kHz. Representative ion mobility spectra for the [y58–2]5+ fragment ion are shown in Figure 6.

Figure 6

Figure 6. Ion mobility spectra of the [y58 – 2]5+ fragment ion generated by UV photodissociation from mobility-selected ubiquitin 8+ precursors stored for 50–500 ms (≈50–500 laser pulses, colored traces). Control spectra after 140 V collisional activation in TIMS-2 (black trace) and without trapping (black dashed trace) are included for comparison. The data show that increasing irradiation time increases fragment yield but does not alter their collision cross sections.

High Resolution Image
Download MS PowerPoint Slide
Figure 6 shows that increasing the UV irradiation time increases fragment ion abundances. However, the collision cross sections of the fragment ions remain unchanged. Importantly, gradual shifts toward larger collision cross sections are not observed even at the longest irradiation time of 500 ms. The absence of irradiation-time–dependent changes in fragment ion collision cross sections indicates that increasing photon exposure enhances cleavage probability but does not induce cumulative vibrational heating sufficient to promote structural annealing of either precursor or fragment ions.
These findings are inconsistent with a mechanism dominated by gradual vibrational heating through repeated photon absorption and internal conversion, which is known to promote CID-type fragmentation. (50) Instead, the data support a scenario in which cleavage occurs primarily from the electronically excited state, while excess vibrational energy is efficiently dissipated by collisional cooling under the high-pressure conditions of the experiment.

Energy Redistribution, Collisional Energy Transfer, and Fragment Ion Conformational Dynamics

The observation of metastable fragment ions in the previous section underlines that UVPD in Tandem-TIMS can produce fragment ions that lack sufficient internal energy to anneal into energetically favored gas-phase structures on the experimental time scale.
We rationalize our results by the much higher gas pressures (∼4 mbar vs 10–5 mbar) and substantially lower UV laser pulse energies (∼10 μJ vs ≥ 1 mJ) than UVPD in typical tandem mass spectrometers. (1,51) Under our low-energy, high-pressure conditions, the most plausible explanation for the observed metastability is that photon absorption deposits insufficient internal energy into the ions to drive gas-phase annealing before collisional vibrational–translational energy transfer dissipates the excess energy into the buffer gas.
To investigate the balance between intramolecular vibrational energy redistribution following UV photon absorption, collisional vibrational–translational energy transfer into the N2 bath gas, and the conformational dynamics of an energetically activated polypeptide, we performed Langevin dynamics simulations. Simulations were carried out on y404+ with an initial excess vibrational energy of 8.5 eV, corresponding to 1.5 times the energy of a 213 nm photon, in a nitrogen gas heat bath at ∼4 mbar (see Figure 7A and Computational Details). The rationale here is that conformational changes would be more likely observed with a higher energetic activation and a reduced size of the polypeptide. The excess energy was initially localized in the Pro1 residue to emulate the localized absorption of the photon at the Pro residue and then allowed to propagate through all vibrational modes of the ion, and, ultimately, to dissipate into the buffer gas via collisions with N2 particles.

Figure 7

Figure 7. Langevin dynamics simulations of vibrational energy redistribution, collisional cooling, and structural dynamics of an energetically activated fragment ion. (A) Simulation setup for the y404+ fragment (cyan) with 8.5 eV of excess vibrational energy initially localized at Pro1. The peptide is immersed in a N2 heat bath (red, ∼4 mbar) in a 120 nm × 120 nm × 120 nm cubic box under periodic boundary conditions. (B) Time evolution of the polypeptide vibrational temperature, T(t), showing that intramolecular vibrational energy redistribution completes within ∼100 ps and raises the temperature to ∼350 K. This is followed by collisional cooling that is well described by a single-exponential decay with τ ≈ 680 ns and equilibration with the 300 K bath within a few microseconds. (C) Comparison of the initial (black) and final (blue, 2.75 μs) polypeptide structures shows that >80% of heavy-atom contacts are maintained, indicating some rearrangement in the loop region but no major global restructuring during gas-phase relaxation.

High Resolution Image
Download MS PowerPoint Slide
The simulations show that this excess vibrational energy, initially confined to Pro1, redistributes over all vibrational modes of the polypeptide within ∼100 ps (Figure 7B). By the time intramolecular vibrational energy redistribution is complete, the effective fragment temperature has reached ∼350 K. Given that the mean time between collisions at these gas pressures is ∼3 ns, this intramolecular redistribution of the photon energy proceeds essentially independently of collisional effects.
The simulations further show that the excess vibrational energy of the fragment ion dissipates into the nitrogen buffer gas via ion–neutral collisions. The time evolution of the vibrational temperature of the polypeptide, T(t), is well described by a single-exponential decay, T(t) ∝ e–t/τ, with a relaxation time constant τ of ∼680 ns (Figure 7B). Under these conditions, the vibrational energy of the polypeptide equilibrates with the 300 K buffer gas within a few microseconds (∼3 μs; ∼1000 collisions, see Figure 7B).
To assess whether transient heating to 350 K is sufficient to induce structural changes in the polypetide, we calculated the fraction of the initial contacts between heavy atoms that are maintained throughout the simulation. Our analysis indicates that >80% of the contacts present in the initial peptide structure are maintained throughout the energy-dissipation process. Figure 7C compares the initial and final polypeptide structures sampled in the simulations, showing that although some rearrangements occur during gas-phase relaxation, the ion internal energy is insufficient to induce global restructuring of the polypeptide before thermalization to the temperature of the buffer gas.

Conclusions

Click to copy section linkSection link copied!
UVPD of proteins has been shown to produce conformation-dependent fragmentation patterns, (20,21) yet direct experimental evidence for a structural relationship between the precursor and the produced fragment ions has been lacking.
Here, using tandem trapped-ion mobility spectrometry/tandem-mass spectrometry, we directly compared the collision cross sections of fragment ions generated from distinct ubiquitin precursor conformations. Our results show:
1)

UVPD in Tandem-TIMS carried out with low-energy UV laser pulse energies (10 μJ) at elevated pressures (∼4 mbar) produces primarily [b+2] and [y-2] type ions at proline residues. Prior reports (16,48) have established these fragment types as markers of direct bond cleavage from the photoexcited electronic state.

2)

These ‘direct-cleavage’ ions produced by UVPD in Tandem-TIMS preserve a structural relationship between precursor and fragment ions: UVPD of the compact, native-like ubiquitin precursor yields fragment ions that maintain a compact structure, with collision cross sections roughly 20% smaller than the equivalent fragments generated from the extended, gas-phase precursor or ions produced by CID.

3)

The compact “direct-cleavage” ions produced by UVPD from the native-like ubiquitin are kinetically trapped in a metastable conformation, with a substantial energy barrier preventing their annealing into energetically favored gas-phase conformations.

The observation of metastable UVPD fragment ions experimentally establishes that aspects of the protein precursor structure may survive backbone dissociation. This finding has potentially important implications for structural mass spectrometry, as it indicates that, under appropriate conditions, fragment ions may provide direct insight into their precursor protein structure.
We rationalize our observations by the combination of the low photon density at our laser pulse energies (∼10 μJ) and the high buffer-gas pressure (∼4 mbar). Because of the low photon flux associated with these laser pulse energies, estimated molecular extinctions coefficients (52) suggest that an individual ubiquitin ion would absorb no more than one or two photons per laser pulse. Absorption of a 213 nm photon at the amide bond chromophore, primarily at proline residues, (16) promotes the ion to an electronically excited state. If the excited state undergoes nonradiative deactivation to the electronic ground state, e.g. via a conical intersection, (53) the resulting vibrationally excited electronic ground state experiences intramolecular vibrational energy redistribution, which raises its temperature by only ∼20–50 K. This is far below the energies required to reach CID-like transition states (>20–30 kcal·mol–1). (54,55) Further, under the high-pressure conditions used here, this modest excess energy is fully dissipated within a few microseconds, so that each laser pulse interacts with ions that have re-equilibrated to the buffer gas temperature. These considerations strongly favor direct dissociation from the photoexcited state as the dominant pathway in our high-pressure, low-photon UVPD setup.
How closely do the structures of these fragment ions reflect those of the precursor? To what extent are native contacts maintained, and how broadly do these findings generalize across other charge states and other proteins? A comprehensive answer to these questions is beyond the scope of the present work and will be addressed in future studies. However, we note that work from both the Brodbelt (18) and Barran (22) laboratories has shown that UVPD of holo-myoglobin preserves the noncovalent interaction between the heme group and the polypeptide in fragments containing the heme-proximal ligand His93. These studies suggest that native tertiary contacts can potentially be maintained in UVPD fragment ions even under substantially lower-pressure conditions, where collisional energy dissipation is less favored for structural preservation than in our high-pressure Tandem-TIMS setup. In this context, the combined experimental and literature evidence renders it plausible that the metastable fragments described here may retain some tertiary contacts and structural aspects of their precursor conformations.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.5c00432.

  • Seven figures with details on tandem-TIMS measurements (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
    • Christian Bleiholder - Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States;  Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32304, United States;  Orcidhttps://orcid.org/0000-0002-4211-1388;  Email: [email protected]
  • Authors
    • Fanny C. Liu - Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States;  Orcidhttps://orcid.org/0000-0003-1403-7114
    • Jusung Lee - Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States
    • Kaira A. Mayberry - Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States
    • Mark E. Ridgeway - Bruker Daltonics, Billerica, Massachusetts 01821, United States
    • Christopher A. Wootton - Bruker Daltonics, Bremen 28359, Germany
    • Alina Theisen - Bruker Daltonics, Bremen 28359, Germany
    • Erin M. Panczyk - Bruker Daltonics, Billerica, Massachusetts 01821, United States;  Orcidhttps://orcid.org/0000-0003-3779-6738
    • Benjamin J. Jones - Bruker Daltonics, Billerica, Massachusetts 01821, United States
    • Lea Nienhaus - Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States;  Orcidhttps://orcid.org/0000-0003-1412-412X
    • Melvin A. Park - Bruker Switzerland AG, Industriestrasse 26, Fällanden 8117, Switzerland;  Orcidhttps://orcid.org/0009-0004-1448-2896
  • Author Contributions

    All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

This research was supported by the National Institutes of Health under award R01GM135682 (C.B.) and the National Science Foundation under grant CHE-2305173 (C.B. and F.C.L.). L.N. acknowledges support from a Camille Dreyfus Teacher-Scholar Award (TC-23-050). K.A.M. acknowledges support from the American Chemical Society Bridge Program.

References

Click to copy section linkSection link copied!

This article references 55 other publications.

  1. 1
    Brodbelt, J. S. Photodissociation Mass Spectrometry: New Tools for Characterization of Biological Molecules. Chem. Soc. Rev. 2014, 43 (8), 2757– 2783,  DOI: 10.1039/C3CS60444F
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Christofi, E.; Barran, P. Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section. Chem. Rev. 2023, 123 (6), 2902– 2949,  DOI: 10.1021/acs.chemrev.2c00600
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J.-E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Amyloid-β Protein Oligomerization and the Importance of Tetramers and Dodecamers in the Aetiology of Alzheimer’s Disease. Nat. Chem. 2009, 1 (4), 326– 331,  DOI: 10.1038/nchem.247
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Bleiholder, C.; Dupuis, N. F.; Wyttenbach, T.; Bowers, M. T. Ion Mobility–Mass Spectrometry Reveals a Conformational Conversion from Random Assembly to β-Sheet in Amyloid Fibril Formation. Nat. Chem. 2011, 3 (2), 172– 177,  DOI: 10.1038/nchem.945
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Zhou, M.; Politis, A.; Davies, R. B.; Liko, I.; Wu, K.-J.; Stewart, A. G.; Stock, D.; Robinson, C. V. Ion Mobility–Mass Spectrometry of a Rotary ATPase Reveals ATP-Induced Reduction in Conformational Flexibility. Nat. Chem. 2014, 6 (3), 208– 215,  DOI: 10.1038/nchem.1868
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Beach, H.; Cole, R.; Gill, M. L.; Loria, J. P. Conservation of Μs–ms Enzyme Motions in the Apo- and Substrate-Mimicked State. J. Am. Chem. Soc. 2005, 127 (25), 9167– 9176,  DOI: 10.1021/ja0514949
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Henzler-Wildman, K. A.; Lei, M.; Thai, V.; Kerns, S. J.; Karplus, M.; Kern, D. A Hierarchy of Timescales in Protein Dynamics Is Linked to Enzyme Catalysis. Nature 2007, 450 (7171), 913– 916,  DOI: 10.1038/nature06407
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Tzeng, S.-R.; Kalodimos, C. G. Dynamic Activation of an Allosteric Regulatory Protein. Nature 2009, 462 (7271), 368– 372,  DOI: 10.1038/nature08560
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Marchand, A.; Czar, M. F.; Eggel, E. N.; Kaeslin, J.; Zenobi, R. Studying Biomolecular Folding and Binding Using Temperature-Jump Mass Spectrometry. Nat. Commun. 2020, 11 (1), 566,  DOI: 10.1038/s41467-019-14179-x
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    McLuckey, S. A.; Goeringer, D. E. Slow Heating Methods in Tandem Mass Spectrometry. J. Mass Spectrom. 1997, 32, 461– 474,  DOI: 10.1002/(SICI)1096-9888(199705)32:5<461::AID-JMS515>3.0.CO;2-H
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Breuker, K.; Oh, H.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation. J. Am. Chem. Soc. 2002, 124 (22), 6407– 6420,  DOI: 10.1021/ja012267j
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Stiving, A. Q.; VanAernum, Z. L.; Busch, F.; Harvey, S. R.; Sarni, S. H.; Wysocki, V. H. Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure. Anal. Chem. 2019, 91 (1), 190– 209,  DOI: 10.1021/acs.analchem.8b05071
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation. J. Am. Chem. Soc. 2013, 135 (34), 12646– 12651,  DOI: 10.1021/ja4029654
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Cammarata, M. B.; Thyer, R.; Rosenberg, J.; Ellington, A.; Brodbelt, J. S. Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry. J. Am. Chem. Soc. 2015, 137 (28), 9128– 9135,  DOI: 10.1021/jacs.5b04628
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Morrison, L. J.; Brodbelt, J. S. 193 Nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology. J. Am. Chem. Soc. 2016, 138 (34), 10849– 10859,  DOI: 10.1021/jacs.6b03905
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Girod, M.; Sanader, Z.; Vojkovic, M.; Antoine, R.; MacAleese, L.; Lemoine, J.; Bonacic-Koutecky, V.; Dugourd, P. UV Photodissociation of Proline-Containing Peptide Ions: Insights from Molecular Dynamics. J. Am. Soc. Mass Spectrom. 2015, 26 (3), 432– 443,  DOI: 10.1007/s13361-014-1038-1
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Zhang, L.; Reilly, J. P. Peptide Photodissociation with 157 Nm Light in a Commercial Tandem Time-of-Flight Mass Spectrometer. Anal. Chem. 2009, 81 (18), 7829– 7838,  DOI: 10.1021/ac9012557
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Cammarata, M. B.; Brodbelt, J. S. Structural Characterization of Holo- and Apo-Myoglobin in the Gas Phase by Ultraviolet Photodissociation Mass Spectrometry. Chem. Sci. 2015, 6 (2), 1324– 1333,  DOI: 10.1039/C4SC03200D
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Cammarata, M.; Thyer, R.; Lombardo, M.; Anderson, A.; Wright, D.; Ellington, A.; Brodbelt, J. S. Characterization of Trimethoprim Resistant E. Coli Dihydrofolate Reductase Mutants by Mass Spectrometry and Inhibition by Propargyl-Linked Antifolates. Chem. Sci. 2017, 8 (5), 4062– 4072,  DOI: 10.1039/C6SC05235E
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Butalewicz, J. P.; Escobar, E. E.; Wootton, C. A.; Theisen, A.; Park, M. A.; Seeley, E. H.; Brodbelt, J. S. Conformational Characterization of Peptides and Proteins by 193 Nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer. Anal. Chem. 2024, 96 (41), 16154– 16161,  DOI: 10.1021/acs.analchem.4c02686
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Butalewicz, J. P.; Sanders, J. D.; Juetten, K. J.; Buzitis, N. W.; Clowers, B. H.; Brodbelt, J. S. Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization. Anal. Chem. 2024, 96 (39), 15674– 15681,  DOI: 10.1021/acs.analchem.4c03119
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Theisen, A.; Black, R.; Corinti, D.; Brown, J. M.; Bellina, B.; Barran, P. E. Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 Nm UVPD Coupled to IM-MS. J. Am. Soc. Mass Spectrom. 2019, 30 (1), 24– 33,  DOI: 10.1007/s13361-018-1992-0
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Liu, F. C.; Ridgeway, M. E.; Winfred, J. S. R. V.; Polfer, N. C.; Lee, J.; Theisen, A.; Wootton, C. A.; Park, M. A.; Bleiholder, C. Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry Coupled with Ultraviolet Photodissociation. Rapid Commun. Mass Spectrom. 2021, 35, e9192  DOI: 10.1002/rcm.9192
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Liu, F. C.; Ridgeway, M. E.; Park, M. A.; Bleiholder, C. Tandem Trapped Ion Mobility Spectrometry. Analyst 2018, 143 (10), 2249– 2258,  DOI: 10.1039/C7AN02054F
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Vijay-Kumar, S.; Bugg, C. E.; Wilkinson, K. D.; Cook, W. J. Three-Dimensional Structure of Ubiquitin at 2.8 A Resolution. Proc. Natl. Acad. Sci. U. S. A. 1985, 82 (11), 3582– 3585,  DOI: 10.1073/pnas.82.11.3582
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    Lange, O. F.; Lakomek, N.-A.; Farès, C.; Schröder, G. F.; Walter, K. F. A.; Becker, S.; Meiler, J.; Grubmüller, H.; Griesinger, C.; de Groot, B. L. Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution. Science 2008, 320 (5882), 1471– 1475,  DOI: 10.1126/science.1157092
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Jourdan, M.; Searle, M. S. Insights into the Stability of Native and Partially Folded States of Ubiquitin: Effects of Cosolvents and Denaturants on the Thermodynamics of Protein Folding. Biochemistry 2001, 40 (34), 10317– 10325,  DOI: 10.1021/bi010767j
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Chai, M.; Young, M. N.; Liu, F. C.; Bleiholder, C. A Transferable, Sample-Independent Calibration Procedure for Trapped Ion Mobility Spectrometry (TIMS). Anal. Chem. 2018, 90 (15), 9040– 9047,  DOI: 10.1021/acs.analchem.8b01326
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Hernandez, D. R.; DeBord, J. D.; Ridgeway, M. E.; Kaplan, D. A.; Park, M. A.; Fernandez-Lima, F. Ion Dynamics in a Trapped Ion Mobility Spectrometer. Analyst 2014, 139 (8), 1913– 1921,  DOI: 10.1039/C3AN02174B
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Liu, F. C.; Ridgeway, M. E.; Wootton, C. A.; Theisen, A.; Panczyk, E. M.; Meier, F.; Park, M. A.; Bleiholder, C. Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis. J. Am. Soc. Mass Spectrom. 2023, 34 (10), 2232– 2246,  DOI: 10.1021/jasms.3c00187
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A. Note: Integration of Trapped Ion Mobility Spectrometry with Mass Spectrometry. Rev. Sci. Instrum. 2011, 82 (12), 126106,  DOI: 10.1063/1.3665933
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Liu, F. C.; Kirk, S. R.; Bleiholder, C. On the Structural Denaturation of Biological Analytes in Trapped Ion Mobility Spectrometry – Mass Spectrometry. Analyst 2016, 141 (12), 3722– 3730,  DOI: 10.1039/C5AN02399H
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Liu, F. C.; Cropley, T. C.; Ridgeway, M. E.; Park, M. A.; Bleiholder, C. Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry–Mass Spectrometry (Tandem-TIMS/MS). Anal. Chem. 2020, 92 (6), 4459– 4467,  DOI: 10.1021/acs.analchem.9b05481
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Cropley, T. C.; Liu, F. C.; Chai, M.; Bush, M. F.; Bleiholder, C. Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior. J. Am. Chem. Soc. 2024, 146, 11115– 11125,  DOI: 10.1021/jacs.3c12892
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Borotto, N. B.; Graham, K. A. Fragmentation and Mobility Separation of Peptide and Protein Ions in a Trapped-Ion Mobility Device. Anal. Chem. 2021, 93 (29), 9959– 9964,  DOI: 10.1021/acs.analchem.1c01188
    Google Scholar
    There is no corresponding record for this reference.
  36. 36
    Silveira, J. A.; Ridgeway, M. E.; Park, M. A. High Resolution Trapped Ion Mobility Spectrometery of Peptides. Anal. Chem. 2014, 86 (12), 5624– 5627,  DOI: 10.1021/ac501261h
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Stow, S. M.; Causon, T. J.; Zheng, X.; Kurulugama, R. T.; Mairinger, T.; May, J. C.; Rennie, E. E.; Baker, E. S.; Smith, R. D.; McLean, J. A.; Hann, S.; Fjeldsted, J. C. An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements. Anal. Chem. 2017, 89 (17), 9048– 9055,  DOI: 10.1021/acs.analchem.7b01729
    Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Loos, M.; Gerber, C.; Corona, F.; Hollender, J.; Singer, H. Accelerated Isotope Fine Structure Calculation Using Pruned Transition Trees. Anal. Chem. 2015, 87 (11), 5738– 5744,  DOI: 10.1021/acs.analchem.5b00941
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. GROMACS: A Message-Passing Parallel Molecular Dynamics Implementation. Comput. Phys. Commun. 1995, 91 (1–3), 43– 56,  DOI: 10.1016/0010-4655(95)00042-E
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. J. Phys. Chem. B 2001, 105 (28), 6474– 6487,  DOI: 10.1021/jp003919d
    Google Scholar
    There is no corresponding record for this reference.
  41. 41
    Wang, S.; Hou, K.; Heinz, H. Accurate and Compatible Force Fields for Molecular Oxygen, Nitrogen, and Hydrogen to Simulate Gases, Electrolytes, and Heterogeneous Interfaces. J. Chem. Theory Comput. 2021, 17 (8), 5198– 5213,  DOI: 10.1021/acs.jctc.0c01132
    Google Scholar
    There is no corresponding record for this reference.
  42. 42
    Wyttenbach, T.; Bowers, M. T. Structural Stability from Solution to the Gas Phase: Native Solution Structure of Ubiquitin Survives Analysis in a Solvent-Free Ion Mobility–Mass Spectrometry Environment. J. Phys. Chem. B 2011, 115 (42), 12266– 12275,  DOI: 10.1021/jp206867a
    Google Scholar
    There is no corresponding record for this reference.
  43. 43
    Laszlo, K. J.; Munger, E. B.; Bush, M. F. Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions. J. Am. Chem. Soc. 2016, 138 (30), 9581– 9588,  DOI: 10.1021/jacs.6b04282
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Warnke, S.; Von Helden, G.; Pagel, K. Protein Structure in the Gas Phase: The Influence of Side-Chain Microsolvation. J. Am. Chem. Soc. 2013, 135 (4), 1177– 1180,  DOI: 10.1021/ja308528d
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Liu, F. C.; Kirk, S. R.; Caldwell, K. A.; Pedrete, T.; Meier, F.; Bleiholder, C. Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections. Anal. Chem. 2022, 94 (23), 8146– 8155,  DOI: 10.1021/acs.analchem.1c05171
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Warnke, S.; Baldauf, C.; Bowers, M. T.; Pagel, K.; von Helden, G. Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds. J. Am. Chem. Soc. 2014, 136 (29), 10308– 10314,  DOI: 10.1021/ja502994b
    Google Scholar
    There is no corresponding record for this reference.
  47. 47
    Fornelli, L.; Srzentić, K.; Toby, T. K.; Doubleday, P. F.; Huguet, R.; Mullen, C.; Melani, R. D.; dos Santos Seckler, H.; DeHart, C. J.; Weisbrod, C. R.; Durbin, K. R.; Greer, J. B.; Early, B. P.; Fellers, R. T.; Zabrouskov, V.; Thomas, P. M.; Compton, P. D.; Kelleher, N. L. Thorough Performance Evaluation of 213 Nm Ultraviolet Photodissociation for Top-down Proteomics. Mol. Cell. Proteomics 2020, 19 (2), 405– 420,  DOI: 10.1074/mcp.TIR119.001638
    Google Scholar
    There is no corresponding record for this reference.
  48. 48
    Devakumar, A.; Thompson, M. S.; Reilly, J. P. Fragmentation of Oligosaccharide Ions with 157 Nm Vacuum Ultraviolet Light. Rapid Commun. Mass Spectrom. 2005, 19 (16), 2313– 2320,  DOI: 10.1002/rcm.2058
    Google Scholar
    There is no corresponding record for this reference.
  49. 49
    Shelimov, K. B.; Jarrold, M. F. Conformations, Unfolding, and Refolding of Apomyoglobin in Vacuum: An Activation Barrier for Gas-Phase Protein Folding. J. Am. Chem. Soc. 1997, 119 (13), 2987– 2994,  DOI: 10.1021/ja962914k
    Google Scholar
    There is no corresponding record for this reference.
  50. 50
    Julian, R. R. The Mechanism Behind Top-Down UVPD Experiments: Making Sense of Apparent Contradictions. J. Am. Soc. Mass Spectrom. 2017, 28 (9), 1823– 1826,  DOI: 10.1007/s13361-017-1721-0
    Google Scholar
    There is no corresponding record for this reference.
  51. 51
    Brodbelt, J. S.; Morrison, L. J.; Santos, I. Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules. Chem. Rev. 2020, 120 (7), 3328– 3380,  DOI: 10.1021/acs.chemrev.9b00440
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Kuipers, B. J. H.; Gruppen, H. Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 Nm To Enable Quantitative Reverse Phase High-Performance Liquid Chromatography–Mass Spectrometry Analysis. J. Agric. Food Chem. 2007, 55 (14), 5445– 5451,  DOI: 10.1021/jf070337l
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Grégoire, G.; Jouvet, C.; Dedonder, C.; Sobolewski, A. L. Ab Initio Study of the Excited-State Deactivation Pathways of Protonated Tryptophan and Tyrosine. J. Am. Chem. Soc. 2007, 129 (19), 6223– 6231,  DOI: 10.1021/ja069050f
    Google Scholar
    There is no corresponding record for this reference.
  54. 54
    Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24 (4), 508– 548,  DOI: 10.1002/mas.20024
    Google Scholar
    There is no corresponding record for this reference.
  55. 55
    Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. Sequence-Scrambling Fragmentation Pathways of Protonated Peptides. J. Am. Chem. Soc. 2008, 130 (52), 17774– 17789,  DOI: 10.1021/ja805074d
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Go to
Get e-Alerts
Get e-Alerts

Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2026, 37, 4, 910–918
Click to copy citationCitation copied!
https://doi.org/10.1021/jasms.5c00432
Published March 11, 2026

Copyright © 2026 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved. This publication is licensed under these Terms of Use.

Request reuse permissions

Article Views

666

Altmetric

-

Citations

-
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. (A) NMR solution structure of the model protein ubiquitin used in this study. (B) Schematic of the tandem-trapped ion mobility spectrometer/tandem-mass spectrometer coupling two trapped ion mobility spectrometers (TIMS-1, blue; TIMS-2, red), an ion trap for ion storage, and a UV laser for photodissociation of the stored ions (green). This platform allows characterizing the precursor protein conformations, performing UVPD of a selected precursor conformation, and subsequently characterizing the structures of the produced fragment ions.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Conformationally selective UVPD of ubiquitin in Tandem-TIMS. (A-i) Native ion mobility/mass spectrometry of ubiquitin showing charge states 7+ and 8+. (ii) Charge state 7+ exhibits mainly a native-like conformer (blue), whereas (iii) charge state 8+ shows a dominant native-like conformation (1283 Å2, blue) and a minor gas-phase structure (1947 Å2, orange). (B-i) Conformationally selective top-down mass spectrometry of ubiquitin charge state 8+. (ii) CID produces typical a-, b-, and y-type fragments, whereas UVPD of the (iii) native-like and (iv) gas-phase ubiquitin conformations shows formation of [a + 2], [b + 2], and [y – 2] fragment ions.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. y585+and [y58 – 2]5+ fragment ion isotope patterns under various measurement conditions. (A) CID in the TIMS-1/TIMS-2 interface produces y585+ ions exclusively. (B, C) UVPD of the compact and extended conformations produces [y58 – 2]5+ fragment ions but no y585+ ions. (D, E) The [y58 – 2]5+ fragment ions are not observed if the ions (D) are not irradiated by UV photons or (E) are irradiated without storage in the ion trap.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. (A) Ion mobility spectra of CID fragment ions produced from the compact (blue) and extended (orange) ubiquitin conformations agree closely. Hence, the produced fragment ions adopt the same gas-phase favored structures, irrespective of the precursor conformation. (B) Ion mobility spectra for the equivalent fragment ions produced by UVPD from the native-like conformation (blue) differ strongly from those produced from the extended, gas-phase conformation (orange). Here, a compact precursor produces fragments with compact conformations, whereas an extended conformation yields fragments with extended structures. This shows that UVPD can generate fragment ions that exist in a nonannealed, metastable conformation.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. Compact UVPD fragment ions produced from the native-like ubiquitin conformation are metastable. The compact (A) [y40 – 2]4+ and (B) [y58 – 2]5+ ions produced by UVPD from the native-like ubiquitin conformation require substantial activation to overcome an energy barrier and unfold into gas-phase favored conformations. The cross sections of the unfolded, gas-phase conformations are consistent with those of the extended fragment ions produced by UVPD from the extended gas-phase ubiquitin conformation and by CID.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. Ion mobility spectra of the [y58 – 2]5+ fragment ion generated by UV photodissociation from mobility-selected ubiquitin 8+ precursors stored for 50–500 ms (≈50–500 laser pulses, colored traces). Control spectra after 140 V collisional activation in TIMS-2 (black trace) and without trapping (black dashed trace) are included for comparison. The data show that increasing irradiation time increases fragment yield but does not alter their collision cross sections.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 7

    Figure 7. Langevin dynamics simulations of vibrational energy redistribution, collisional cooling, and structural dynamics of an energetically activated fragment ion. (A) Simulation setup for the y404+ fragment (cyan) with 8.5 eV of excess vibrational energy initially localized at Pro1. The peptide is immersed in a N2 heat bath (red, ∼4 mbar) in a 120 nm × 120 nm × 120 nm cubic box under periodic boundary conditions. (B) Time evolution of the polypeptide vibrational temperature, T(t), showing that intramolecular vibrational energy redistribution completes within ∼100 ps and raises the temperature to ∼350 K. This is followed by collisional cooling that is well described by a single-exponential decay with τ ≈ 680 ns and equilibration with the 300 K bath within a few microseconds. (C) Comparison of the initial (black) and final (blue, 2.75 μs) polypeptide structures shows that >80% of heavy-atom contacts are maintained, indicating some rearrangement in the loop region but no major global restructuring during gas-phase relaxation.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 55 other publications.

    1. 1
      Brodbelt, J. S. Photodissociation Mass Spectrometry: New Tools for Characterization of Biological Molecules. Chem. Soc. Rev. 2014, 43 (8), 2757– 2783,  DOI: 10.1039/C3CS60444F
      There is no corresponding record for this reference.
    2. 2
      Christofi, E.; Barran, P. Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section. Chem. Rev. 2023, 123 (6), 2902– 2949,  DOI: 10.1021/acs.chemrev.2c00600
      There is no corresponding record for this reference.
    3. 3
      Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J.-E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Amyloid-β Protein Oligomerization and the Importance of Tetramers and Dodecamers in the Aetiology of Alzheimer’s Disease. Nat. Chem. 2009, 1 (4), 326– 331,  DOI: 10.1038/nchem.247
      There is no corresponding record for this reference.
    4. 4
      Bleiholder, C.; Dupuis, N. F.; Wyttenbach, T.; Bowers, M. T. Ion Mobility–Mass Spectrometry Reveals a Conformational Conversion from Random Assembly to β-Sheet in Amyloid Fibril Formation. Nat. Chem. 2011, 3 (2), 172– 177,  DOI: 10.1038/nchem.945
      There is no corresponding record for this reference.
    5. 5
      Zhou, M.; Politis, A.; Davies, R. B.; Liko, I.; Wu, K.-J.; Stewart, A. G.; Stock, D.; Robinson, C. V. Ion Mobility–Mass Spectrometry of a Rotary ATPase Reveals ATP-Induced Reduction in Conformational Flexibility. Nat. Chem. 2014, 6 (3), 208– 215,  DOI: 10.1038/nchem.1868
      There is no corresponding record for this reference.
    6. 6
      Beach, H.; Cole, R.; Gill, M. L.; Loria, J. P. Conservation of Μs–ms Enzyme Motions in the Apo- and Substrate-Mimicked State. J. Am. Chem. Soc. 2005, 127 (25), 9167– 9176,  DOI: 10.1021/ja0514949
      There is no corresponding record for this reference.
    7. 7
      Henzler-Wildman, K. A.; Lei, M.; Thai, V.; Kerns, S. J.; Karplus, M.; Kern, D. A Hierarchy of Timescales in Protein Dynamics Is Linked to Enzyme Catalysis. Nature 2007, 450 (7171), 913– 916,  DOI: 10.1038/nature06407
      There is no corresponding record for this reference.
    8. 8
      Tzeng, S.-R.; Kalodimos, C. G. Dynamic Activation of an Allosteric Regulatory Protein. Nature 2009, 462 (7271), 368– 372,  DOI: 10.1038/nature08560
      There is no corresponding record for this reference.
    9. 9
      Marchand, A.; Czar, M. F.; Eggel, E. N.; Kaeslin, J.; Zenobi, R. Studying Biomolecular Folding and Binding Using Temperature-Jump Mass Spectrometry. Nat. Commun. 2020, 11 (1), 566,  DOI: 10.1038/s41467-019-14179-x
      There is no corresponding record for this reference.
    10. 10
      McLuckey, S. A.; Goeringer, D. E. Slow Heating Methods in Tandem Mass Spectrometry. J. Mass Spectrom. 1997, 32, 461– 474,  DOI: 10.1002/(SICI)1096-9888(199705)32:5<461::AID-JMS515>3.0.CO;2-H
      There is no corresponding record for this reference.
    11. 11
      Breuker, K.; Oh, H.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation. J. Am. Chem. Soc. 2002, 124 (22), 6407– 6420,  DOI: 10.1021/ja012267j
      There is no corresponding record for this reference.
    12. 12
      Stiving, A. Q.; VanAernum, Z. L.; Busch, F.; Harvey, S. R.; Sarni, S. H.; Wysocki, V. H. Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure. Anal. Chem. 2019, 91 (1), 190– 209,  DOI: 10.1021/acs.analchem.8b05071
      There is no corresponding record for this reference.
    13. 13
      Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation. J. Am. Chem. Soc. 2013, 135 (34), 12646– 12651,  DOI: 10.1021/ja4029654
      There is no corresponding record for this reference.
    14. 14
      Cammarata, M. B.; Thyer, R.; Rosenberg, J.; Ellington, A.; Brodbelt, J. S. Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry. J. Am. Chem. Soc. 2015, 137 (28), 9128– 9135,  DOI: 10.1021/jacs.5b04628
      There is no corresponding record for this reference.
    15. 15
      Morrison, L. J.; Brodbelt, J. S. 193 Nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology. J. Am. Chem. Soc. 2016, 138 (34), 10849– 10859,  DOI: 10.1021/jacs.6b03905
      There is no corresponding record for this reference.
    16. 16
      Girod, M.; Sanader, Z.; Vojkovic, M.; Antoine, R.; MacAleese, L.; Lemoine, J.; Bonacic-Koutecky, V.; Dugourd, P. UV Photodissociation of Proline-Containing Peptide Ions: Insights from Molecular Dynamics. J. Am. Soc. Mass Spectrom. 2015, 26 (3), 432– 443,  DOI: 10.1007/s13361-014-1038-1
      There is no corresponding record for this reference.
    17. 17
      Zhang, L.; Reilly, J. P. Peptide Photodissociation with 157 Nm Light in a Commercial Tandem Time-of-Flight Mass Spectrometer. Anal. Chem. 2009, 81 (18), 7829– 7838,  DOI: 10.1021/ac9012557
      There is no corresponding record for this reference.
    18. 18
      Cammarata, M. B.; Brodbelt, J. S. Structural Characterization of Holo- and Apo-Myoglobin in the Gas Phase by Ultraviolet Photodissociation Mass Spectrometry. Chem. Sci. 2015, 6 (2), 1324– 1333,  DOI: 10.1039/C4SC03200D
      There is no corresponding record for this reference.
    19. 19
      Cammarata, M.; Thyer, R.; Lombardo, M.; Anderson, A.; Wright, D.; Ellington, A.; Brodbelt, J. S. Characterization of Trimethoprim Resistant E. Coli Dihydrofolate Reductase Mutants by Mass Spectrometry and Inhibition by Propargyl-Linked Antifolates. Chem. Sci. 2017, 8 (5), 4062– 4072,  DOI: 10.1039/C6SC05235E
      There is no corresponding record for this reference.
    20. 20
      Butalewicz, J. P.; Escobar, E. E.; Wootton, C. A.; Theisen, A.; Park, M. A.; Seeley, E. H.; Brodbelt, J. S. Conformational Characterization of Peptides and Proteins by 193 Nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer. Anal. Chem. 2024, 96 (41), 16154– 16161,  DOI: 10.1021/acs.analchem.4c02686
      There is no corresponding record for this reference.
    21. 21
      Butalewicz, J. P.; Sanders, J. D.; Juetten, K. J.; Buzitis, N. W.; Clowers, B. H.; Brodbelt, J. S. Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization. Anal. Chem. 2024, 96 (39), 15674– 15681,  DOI: 10.1021/acs.analchem.4c03119
      There is no corresponding record for this reference.
    22. 22
      Theisen, A.; Black, R.; Corinti, D.; Brown, J. M.; Bellina, B.; Barran, P. E. Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 Nm UVPD Coupled to IM-MS. J. Am. Soc. Mass Spectrom. 2019, 30 (1), 24– 33,  DOI: 10.1007/s13361-018-1992-0
      There is no corresponding record for this reference.
    23. 23
      Liu, F. C.; Ridgeway, M. E.; Winfred, J. S. R. V.; Polfer, N. C.; Lee, J.; Theisen, A.; Wootton, C. A.; Park, M. A.; Bleiholder, C. Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry Coupled with Ultraviolet Photodissociation. Rapid Commun. Mass Spectrom. 2021, 35, e9192  DOI: 10.1002/rcm.9192
      There is no corresponding record for this reference.
    24. 24
      Liu, F. C.; Ridgeway, M. E.; Park, M. A.; Bleiholder, C. Tandem Trapped Ion Mobility Spectrometry. Analyst 2018, 143 (10), 2249– 2258,  DOI: 10.1039/C7AN02054F
      There is no corresponding record for this reference.
    25. 25
      Vijay-Kumar, S.; Bugg, C. E.; Wilkinson, K. D.; Cook, W. J. Three-Dimensional Structure of Ubiquitin at 2.8 A Resolution. Proc. Natl. Acad. Sci. U. S. A. 1985, 82 (11), 3582– 3585,  DOI: 10.1073/pnas.82.11.3582
      There is no corresponding record for this reference.
    26. 26
      Lange, O. F.; Lakomek, N.-A.; Farès, C.; Schröder, G. F.; Walter, K. F. A.; Becker, S.; Meiler, J.; Grubmüller, H.; Griesinger, C.; de Groot, B. L. Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution. Science 2008, 320 (5882), 1471– 1475,  DOI: 10.1126/science.1157092
      There is no corresponding record for this reference.
    27. 27
      Jourdan, M.; Searle, M. S. Insights into the Stability of Native and Partially Folded States of Ubiquitin: Effects of Cosolvents and Denaturants on the Thermodynamics of Protein Folding. Biochemistry 2001, 40 (34), 10317– 10325,  DOI: 10.1021/bi010767j
      There is no corresponding record for this reference.
    28. 28
      Chai, M.; Young, M. N.; Liu, F. C.; Bleiholder, C. A Transferable, Sample-Independent Calibration Procedure for Trapped Ion Mobility Spectrometry (TIMS). Anal. Chem. 2018, 90 (15), 9040– 9047,  DOI: 10.1021/acs.analchem.8b01326
      There is no corresponding record for this reference.
    29. 29
      Hernandez, D. R.; DeBord, J. D.; Ridgeway, M. E.; Kaplan, D. A.; Park, M. A.; Fernandez-Lima, F. Ion Dynamics in a Trapped Ion Mobility Spectrometer. Analyst 2014, 139 (8), 1913– 1921,  DOI: 10.1039/C3AN02174B
      There is no corresponding record for this reference.
    30. 30
      Liu, F. C.; Ridgeway, M. E.; Wootton, C. A.; Theisen, A.; Panczyk, E. M.; Meier, F.; Park, M. A.; Bleiholder, C. Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis. J. Am. Soc. Mass Spectrom. 2023, 34 (10), 2232– 2246,  DOI: 10.1021/jasms.3c00187
      There is no corresponding record for this reference.
    31. 31
      Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A. Note: Integration of Trapped Ion Mobility Spectrometry with Mass Spectrometry. Rev. Sci. Instrum. 2011, 82 (12), 126106,  DOI: 10.1063/1.3665933
      There is no corresponding record for this reference.
    32. 32
      Liu, F. C.; Kirk, S. R.; Bleiholder, C. On the Structural Denaturation of Biological Analytes in Trapped Ion Mobility Spectrometry – Mass Spectrometry. Analyst 2016, 141 (12), 3722– 3730,  DOI: 10.1039/C5AN02399H
      There is no corresponding record for this reference.
    33. 33
      Liu, F. C.; Cropley, T. C.; Ridgeway, M. E.; Park, M. A.; Bleiholder, C. Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry–Mass Spectrometry (Tandem-TIMS/MS). Anal. Chem. 2020, 92 (6), 4459– 4467,  DOI: 10.1021/acs.analchem.9b05481
      There is no corresponding record for this reference.
    34. 34
      Cropley, T. C.; Liu, F. C.; Chai, M.; Bush, M. F.; Bleiholder, C. Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior. J. Am. Chem. Soc. 2024, 146, 11115– 11125,  DOI: 10.1021/jacs.3c12892
      There is no corresponding record for this reference.
    35. 35
      Borotto, N. B.; Graham, K. A. Fragmentation and Mobility Separation of Peptide and Protein Ions in a Trapped-Ion Mobility Device. Anal. Chem. 2021, 93 (29), 9959– 9964,  DOI: 10.1021/acs.analchem.1c01188
      There is no corresponding record for this reference.
    36. 36
      Silveira, J. A.; Ridgeway, M. E.; Park, M. A. High Resolution Trapped Ion Mobility Spectrometery of Peptides. Anal. Chem. 2014, 86 (12), 5624– 5627,  DOI: 10.1021/ac501261h
      There is no corresponding record for this reference.
    37. 37
      Stow, S. M.; Causon, T. J.; Zheng, X.; Kurulugama, R. T.; Mairinger, T.; May, J. C.; Rennie, E. E.; Baker, E. S.; Smith, R. D.; McLean, J. A.; Hann, S.; Fjeldsted, J. C. An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements. Anal. Chem. 2017, 89 (17), 9048– 9055,  DOI: 10.1021/acs.analchem.7b01729
      There is no corresponding record for this reference.
    38. 38
      Loos, M.; Gerber, C.; Corona, F.; Hollender, J.; Singer, H. Accelerated Isotope Fine Structure Calculation Using Pruned Transition Trees. Anal. Chem. 2015, 87 (11), 5738– 5744,  DOI: 10.1021/acs.analchem.5b00941
      There is no corresponding record for this reference.
    39. 39
      Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. GROMACS: A Message-Passing Parallel Molecular Dynamics Implementation. Comput. Phys. Commun. 1995, 91 (1–3), 43– 56,  DOI: 10.1016/0010-4655(95)00042-E
      There is no corresponding record for this reference.
    40. 40
      Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. J. Phys. Chem. B 2001, 105 (28), 6474– 6487,  DOI: 10.1021/jp003919d
      There is no corresponding record for this reference.
    41. 41
      Wang, S.; Hou, K.; Heinz, H. Accurate and Compatible Force Fields for Molecular Oxygen, Nitrogen, and Hydrogen to Simulate Gases, Electrolytes, and Heterogeneous Interfaces. J. Chem. Theory Comput. 2021, 17 (8), 5198– 5213,  DOI: 10.1021/acs.jctc.0c01132
      There is no corresponding record for this reference.
    42. 42
      Wyttenbach, T.; Bowers, M. T. Structural Stability from Solution to the Gas Phase: Native Solution Structure of Ubiquitin Survives Analysis in a Solvent-Free Ion Mobility–Mass Spectrometry Environment. J. Phys. Chem. B 2011, 115 (42), 12266– 12275,  DOI: 10.1021/jp206867a
      There is no corresponding record for this reference.
    43. 43
      Laszlo, K. J.; Munger, E. B.; Bush, M. F. Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions. J. Am. Chem. Soc. 2016, 138 (30), 9581– 9588,  DOI: 10.1021/jacs.6b04282
      There is no corresponding record for this reference.
    44. 44
      Warnke, S.; Von Helden, G.; Pagel, K. Protein Structure in the Gas Phase: The Influence of Side-Chain Microsolvation. J. Am. Chem. Soc. 2013, 135 (4), 1177– 1180,  DOI: 10.1021/ja308528d
      There is no corresponding record for this reference.
    45. 45
      Liu, F. C.; Kirk, S. R.; Caldwell, K. A.; Pedrete, T.; Meier, F.; Bleiholder, C. Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections. Anal. Chem. 2022, 94 (23), 8146– 8155,  DOI: 10.1021/acs.analchem.1c05171
      There is no corresponding record for this reference.
    46. 46
      Warnke, S.; Baldauf, C.; Bowers, M. T.; Pagel, K.; von Helden, G. Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds. J. Am. Chem. Soc. 2014, 136 (29), 10308– 10314,  DOI: 10.1021/ja502994b
      There is no corresponding record for this reference.
    47. 47
      Fornelli, L.; Srzentić, K.; Toby, T. K.; Doubleday, P. F.; Huguet, R.; Mullen, C.; Melani, R. D.; dos Santos Seckler, H.; DeHart, C. J.; Weisbrod, C. R.; Durbin, K. R.; Greer, J. B.; Early, B. P.; Fellers, R. T.; Zabrouskov, V.; Thomas, P. M.; Compton, P. D.; Kelleher, N. L. Thorough Performance Evaluation of 213 Nm Ultraviolet Photodissociation for Top-down Proteomics. Mol. Cell. Proteomics 2020, 19 (2), 405– 420,  DOI: 10.1074/mcp.TIR119.001638
      There is no corresponding record for this reference.
    48. 48
      Devakumar, A.; Thompson, M. S.; Reilly, J. P. Fragmentation of Oligosaccharide Ions with 157 Nm Vacuum Ultraviolet Light. Rapid Commun. Mass Spectrom. 2005, 19 (16), 2313– 2320,  DOI: 10.1002/rcm.2058
      There is no corresponding record for this reference.
    49. 49
      Shelimov, K. B.; Jarrold, M. F. Conformations, Unfolding, and Refolding of Apomyoglobin in Vacuum: An Activation Barrier for Gas-Phase Protein Folding. J. Am. Chem. Soc. 1997, 119 (13), 2987– 2994,  DOI: 10.1021/ja962914k
      There is no corresponding record for this reference.
    50. 50
      Julian, R. R. The Mechanism Behind Top-Down UVPD Experiments: Making Sense of Apparent Contradictions. J. Am. Soc. Mass Spectrom. 2017, 28 (9), 1823– 1826,  DOI: 10.1007/s13361-017-1721-0
      There is no corresponding record for this reference.
    51. 51
      Brodbelt, J. S.; Morrison, L. J.; Santos, I. Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules. Chem. Rev. 2020, 120 (7), 3328– 3380,  DOI: 10.1021/acs.chemrev.9b00440
      There is no corresponding record for this reference.
    52. 52
      Kuipers, B. J. H.; Gruppen, H. Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 Nm To Enable Quantitative Reverse Phase High-Performance Liquid Chromatography–Mass Spectrometry Analysis. J. Agric. Food Chem. 2007, 55 (14), 5445– 5451,  DOI: 10.1021/jf070337l
      There is no corresponding record for this reference.
    53. 53
      Grégoire, G.; Jouvet, C.; Dedonder, C.; Sobolewski, A. L. Ab Initio Study of the Excited-State Deactivation Pathways of Protonated Tryptophan and Tyrosine. J. Am. Chem. Soc. 2007, 129 (19), 6223– 6231,  DOI: 10.1021/ja069050f
      There is no corresponding record for this reference.
    54. 54
      Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24 (4), 508– 548,  DOI: 10.1002/mas.20024
      There is no corresponding record for this reference.
    55. 55
      Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. Sequence-Scrambling Fragmentation Pathways of Protonated Peptides. J. Am. Chem. Soc. 2008, 130 (52), 17774– 17789,  DOI: 10.1021/ja805074d
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.5c00432.

    • Seven figures with details on tandem-TIMS measurements (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.