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The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.

Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li + , Na + , K + , Rb + , and Cs + ) and halogen anions (Cl - , Br - , and I - ) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li] + molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I] - molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 5 2 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 6 1 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature ( T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å 2 and ∼3250-15,385 Å 2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact β-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N -acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m / z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

There is a growing demand for lower-cost, benchtop analytical instruments with complementary separation capabilities for the screening and characterization of biological samples. In this study, we report on the custom integration of trapped ion mobility spectrometry and ultraviolet photodissociation capabilities in a commercial Paul quadrupolar ion trap multistage mass spectrometer (TIMS-QIT-MS n UVPD platform). A gated TIMS operation allowed for the accumulation of ion mobility separated ion in the QIT, followed by a mass analysis (MS1 scan) or m / z isolation, followed by selected collision induced dissociation (CID) or ultraviolet photodissociation (UVPD) and a mass analysis (MS2 scan). The analytical potential of this platform for the analysis of complex and labile biological samples is illustrated for the case of positional isomers with varying PTM location of the histone H4 tryptic peptide 4-17 singly and doubly acetylated and the histone H3.1 tail (1-50) singly trimethylated. For all cases, a baseline ion mobility precursor molecular ion preseparation was obtained. The tandem CID and UVPD MS2 allowed for effective sequence confirmation as well as the identification of reporter fragment ions associated with the PTM location; a higher sequence coverage was obtained using UVPD when compared to CID. Different from previous IMS-MS implementation, the novel TIMS-QIT-MS n UVPD platform offers a lower-cost alternative for the structural characterization of biological molecules that can be widely disseminated in clinical laboratories.

Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant ( K d ) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c 30 5+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me 2 , 2Ac + Me 2 and 3Ac + Me 2 ), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c 30 5+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA 22 ) and a 50-nt (DNA 50 ) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH 4 Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Trapped ion mobility spectrometry (TIMS) when coupled with mass spectrometry (MS) offers great advantages for the separation of isobaric, isomeric, and/or conformeric species. In the present work, we report the advantages of coupling TIMS with a low-cost, ultraviolet photodissociation (UVPD) linear ion trap operated at few mbars prior to time-of-flight (ToF) MS analysis for the effective characterization of isobaric, isomeric, and/or conformeric species based on mobility-selected fragmentation patterns. These three traditional challenges to MS-based separations are illustrated for the case of biologically relevant model systems: H3.1 histone tail PTM isobars (K4Me3/K18Ac), lanthipeptide regioisomers (overlapping/nonoverlapping ring patterns), and a model peptide conformer (angiotensin I). The sequential nature of the TIMS operation allows for effective synchronization with the ToF MS scans, in addition to parallel operation between the TIMS and the UVPD trap. Inspection of the mobility-selected UVPD MS spectra showed that for all three cases considered, unique fragmentation patterns (fingerprints) were observed per mobility band. Different from other IMS-UVPD implementations, the higher resolution of the TIMS device allowed for high mobility resolving power ( R > 100) and effective mobility separation. The mobility selected UVPD MS provided high sequence coverage (>85%) with a fragmentation efficiency up to ∼40%.

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry-tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

Lasso peptides are members of the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we describe the first lasso peptide originating from a biosynthetic gene cluster belonging to a unique lasso peptide subclade defined by the presence of a bifunctional protein harboring both a leader peptidase (B2) and an ABC transporter (D) domain. Bioinformatic analysis revealed that these clusters also encode homologues of the NisR/NisK regulatory system and the NisF/NisE/NisG immunity factors, which are usually associated with the clusters of antimicrobial class I lanthipeptides, such as nisin, another distinct RiPP subfamily. The cluster enabling the heterologous production of the lasso peptide cochonodin I in E. coli originated from Streptococcus suis LSS65, and the threaded structure of cochonodin I was evidenced through extensive MS/MS analysis and stability assays. It was shown that the ABC transporter domain from SsuB2/D is not essential for lasso peptide maturation. By extensive genome mining dedicated exclusively to other lasso peptide biosynthetic gene clusters featuring bifunctional B2/D proteins, it was furthermore revealed that many bacteria associated with human or animal microbiota hold the biosynthetic potential to produce cochonodin-like lasso peptides, implying that these natural products might play roles in human and animal health.

Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvβ3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded b i and y j fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Comprehensive characterization of post-translationally modified histone proteoforms is challenging due to their high isobaric and isomeric content. Trapped ion mobility spectrometry (TIMS), implemented on a quadrupole/time-of-flight (Q-ToF) mass spectrometer, has shown great promise in discriminating isomeric complete histone tails. The absence of electron activated dissociation (ExD) in the current platform prevents the comprehensive characterization of unknown histone proteoforms. In the present work, we report for the first time the use of an electromagnetostatic (EMS) cell devised for nonergodic dissociation based on electron capture dissociation (ECD), implemented within a nESI-TIMS-Q-ToF mass spectrometer for the characterization of acetylated (AcK18 and AcK27) and trimethylated (TriMetK4, TriMetK9 and TriMetK27) complete histone tails. The integration of the EMS cell in a TIMS-q-TOF MS permitted fast mobility-selected top-down ECD fragmentation with near 10% efficiency overall. The potential of this coupling was illustrated using isobaric (AcK18/TriMetK4) and isomeric (AcK18/AcK27 and TriMetK4/TriMetK9) binary H3 histone tail mixtures, and the H3.1 TriMetK27 histone tail structural diversity (e.g., three IMS bands at z = 7+). The binary isobaric and isomeric mixtures can be separated in the mobility domain with R IMS > 100 and the nonergodic ECD fragmentation permitted the PTM localization (sequence coverage of ∼86%). Differences in the ECD patterns per mobility band of the z = 7+ H3 TriMetK27 molecular ions suggested that the charge location is responsible for the structural differences observed in the mobility domain. This coupling further enhances the structural toolbox with fast, high resolution mobility separations in tandem with nonergodic fragmentation for effective proteoform differentiation.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range ( K 0 = 0.185-1.84 cm 2 ·V -1 ·s -1 ), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate ( Sr ) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS N 2 ) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters ( n = 6-73), Tuning Mix oligomers ( n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin ( n = 1-4), carbonic anhydrase, β clamp ( n = 1-4), topoisomerase IB, bovine serum albumin ( n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase ( n = 1-2)) covering a wide mass (up to m / z 19 000) and CCS range (up to 22 000 Å 2 with <0.6% relative standard deviation (RSD)).

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature ( T ∼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H] 12+ to [M + 16H] 16+ at ∼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H] 46+ at ∼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature T m ∼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 μM), this method can detect nonspecific BSA dimers and trimers which dissociate early ( T d ∼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).

Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. BI-32169 is a class III lasso peptide containing one disulfide bond that further stabilizes the lasso structure. In contrast to its branched-cyclic analog, BI-32169 has higher stability and is known to exert a potent inhibitory activity against the human glucagon receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) experiments were carried out to evidence specific structural signatures of the two topologies. CID experiments showed similar fragmentation patterns for the two topoisomers, where a part of the C-terminal tail remains covalently linked to the macrolactam ring by the disulfide bond, which cannot clearly constitute a signature of the lasso topology. ECD experiments of BI-32169 showed an increase of hydrogen migration events in the loop region when compared with those of its branched-cyclic topoisomer evidencing specific structural signatures for the lasso topology. The high mobility resolving power of TIMS resulted in the identification of multiple conformations for the protonated species but did not allow the clear differentiation of the two topologies in mixture. When in complex with cesium metal ions, a reduced number of conformations led to a clear identification of the two structures. Experiments reducing and alkylating the disulfide bond of BI-32169 showed that the lasso structure is preserved and heat stable and the associated conformational changes provide new insights about the role of the disulfide bond in the inhibitory activity against the human glucagon receptor. Graphical abstract ᅟ.

The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli β-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The β L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recently, we proposed a high-throughput screening workflow for the elucidation of agonistic or antagonistic growth hormone-releasing hormone (GHRH) potencies based on structural motif descriptors as a function of the starting solution. In the present work, we revisited the influence of solution and gas-phase GHRH molecular microenvironment using trapped ion mobility-mass spectrometry (TIMS-MS). The effect of the starting solvent composition (10 mM ammonium acetate (NH 4 Ac), 50% methanol (MeOH), 50% acetonitrile (MeCN), and 50% acetone (Ac)) and gas-phase modifiers (N 2 , N 2 + MeOH, N 2 + MeCN, and N 2 + Ac) on the conformational states of three GHRH analogues, GHRH (1-29), MR-406, and MIA-602, is described as a function of the trapping time (100-500 ms). Changes in the mobility profiles were observed showing the dependence of the conformational states of GHRH analogues according to the molecular microenvironment in solution, suggesting the presence of solution memory effects on the gas-phase observed structures. Modifying the bath gas composition resulted in smaller mobilities that are correlated with the size and mass of the organic modifier, and more importantly led to substantial changes in relative abundances of the IMS profiles. We attributed the observed changes in the mobility profiles by a clustering/declustering mechanism between the GHRH analogue ions and the gas modifiers, redefining the free energy landscape and leading to other local minima structures. Moreover, inspection of the mobility profiles as a function of the trapping time (100-500 ms) allowed for conformational interconversions toward more stable "gas-phase" structures. These experiments enabled us to outline a more detailed description of the structures and intermediates involved in the biological activity of GHRH, MR-406, and MIA-602.

Microcin J25 is a ribosomal synthesized and post-translationally modified peptide (RiPP) characterized by a mechanically interlocked topology called the lasso fold. This structure provides microcin J25 a potent antimicrobial activity resulting from internalization via the siderophore receptor FhuA and further inhibition of the RNA polymerase. In the present work, nuclear magnetic resonance (NMR) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate the lasso structure of microcin J25. NMR experiments showed that the lasso peptide microcin J25 can adopt conformational states where Pro16 can be found in the cis- and trans-orientations. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P7A], [P16A], and [P7A/P16A] variants), demonstrated that microcin J25 can adopt cis/cis-, cis/trans-, trans/cis-, and trans/trans-conformations at the Pro7 and Pro16 peptide bonds. It was also shown that interconversion between the conformers can occur as a function of the starting solvent conditions and ion heating (collision-induced activation, CIA) despite the lasso topology. Complementary to NMR findings, the cis-conformations at Pro7 were assigned using TIMS-MS. This study highlights the analytical power of TIMS-MS and site-directed mutagenesis for the study of biological systems with large micro-heterogeneity as a way to further increase our understanding of the receptor-binding dynamics and biological activity.

Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to ∼410 ( R ∼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.

Quantitation of the serum concentration of 25-hydroxyvitamin D is a high-demand assay that suffers from long chromatography time to separate 25-hydroxyvitamin D from its inactive epimer; however, ion mobility spectrometry can distinguish the epimer pair in under 30 ms due to the presence of a unique extended or "open" gas-phase sodiated conformer, not shared with the epimer, reducing the need for chromatographic separation. Five ion mobility mass spectrometers utilizing commercially available IMS technologies, including drift tube, traveling wave, trapped, and high-field asymmetric ion mobility spectrometry, are evaluated for their ability to resolve the unique open conformer. Additionally, settings for each instrument are evaluated to understand their influence on ion heating, which can drive the open conformer into a compact or "closed" conformer shared with the epimer. The four low-field instruments successfully resolved the open conformer from the closed conformer at baseline or near-baseline resolution at typical operating parameters. High-field asymmetric ion mobility was unable to resolve a unique peak but detected two peaks for the epimer, in contrast to the low-field methods that detected one conformer. This study seeks to expand the instrument space by highlighting the potential of each platform for the separation of 25-hydroxyvitamin D epimers.

Topoisomerases are important complex enzymes that modulate DNA topology to maintain chromosome superstructure and integrity. These enzymes are involved in many cellular processes that resolve specific DNA superstructures and intermediates. The low abundance combined with the biological heterogeneity of relevant intermediates of topoisomerases makes their structural information not readily accessible using traditional structural biology tools (e.g., NMR and X-ray crystallography). In the present work, a second-generation trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to study Escherichia coli topoisomerase IA (EcTopIA) and variola virus topoisomerase IB (vTopIB) as well as their complexes with a single-stranded DNA and a stem-loop DNA under native conditions. The higher trapping efficiency and extended mass range of the new, convex TIMS geometry allowed for the separation and identification of multiple conformational states for the two topoisomerases and their DNA complexes. Inspection of the conformational space of EcTopIA and vTopIB in complex with DNA showed that upon DNA binding, the number of conformational states is significantly reduced, suggesting that the DNA binding selects for a narrow range of conformers restricted by the interaction with the DNA substrate. The large microheterogeneity observed for the two DNA binding proteins suggests that they can have multiple biological functions. This work highlights the potential of TIMS-MS for the structural investigations of intrinsically disordered proteins (e.g., DNA binding proteins) as a way to gain a better understanding of the mechanisms involved in DNA substrate recognition, binding, and assembly of the catalytically active enzyme-DNA complex., Competing Interests: The authors declare no competing financial interest.

The synthesis of analogues of hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) is an efficient strategy for designing new therapeutic agents. Several promising synthetic agonist and antagonist analogues of GHRH have been developed based on amino acid mutations of the GHRH (1-29) sequence. Because structural information on the activity of the GHRH agonists or antagonists is limited, there is a need for more effective analytical workflows capable of correlating the peptide sequence with biological activity. In the present work, three GHRH agonists-MR-356, MR-406, and MR-409-and three GHRH antagonists-MIA-602, MIA-606, and MIA-690-were investigated to assess the role of substitutions in the amino acid sequence on structural motifs and receptor binding affinities. The use of high resolution trapped ion mobility spectrometry coupled to mass spectrometry allowed the observation of a large number of peptide-specific mobility bands (or structural motif descriptors) as a function of the amino acid sequence and the starting solution environment. A direct correlation was observed between the amino acid substitutions (i.e., basic residues and d/l-amino acids), the structural motif descriptors, and the biological function (i.e., receptor binding affinities of the GHRH agonists and antagonists). The simplicity, ease, and high throughput of the proposed workflow based on the structural motif descriptors can significantly reduce the cost and time during screening of new synthetic peptide analogues., Competing Interests: The authors declare no competing financial interest.

Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions (r ∼ 2.1 on average). It is also shown that the metal incorporation (e.g., doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS.

Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD + ) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD + and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD + and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a "close" to "open" conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD + protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD + profiles showed no dependence. IRMPD spectroscopy of NAD + and NADH protonated species in the 800-1800 and 3200-3700 cm -1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD + and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD + and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N-H-O and O-H-O forms with ribose rings.

Comprehensive characterization of proteomes comprising the same proteins with distinct post-translational modifications (PTMs) is a staggering challenge. Many such proteoforms are isomers (localization variants) that require separation followed by top-down or middle-down mass spectrometric analyses, but condensed-phase separations are ineffective in those size ranges. The variants for "middle-down" peptides were resolved by differential ion mobility spectrometry (FAIMS), relying on the mobility increment at high electric fields, but not previously by linear IMS on the basis of absolute mobility. We now use complete histone tails with diverse PTMs on alternative sites to demonstrate that high-resolution linear IMS, here trapped IMS (TIMS), broadly resolves the variants of ∼50 residues in full or into binary mixtures quantifiable by tandem MS, largely thanks to orthogonal separations across charge states. Separations using traveling-wave (TWIMS) and/or involving various time scales and electrospray ionization source conditions are similar (with lower resolution for TWIMS), showing the transferability of results across linear IMS instruments. The linear IMS and FAIMS dimensions are substantially orthogonal, suggesting FAIMS/IMS/MS as a powerful platform for proteoform analyses.

Despite often minute concentrations in vivo, d-amino acid containing peptides (DAACPs) are crucial to many life processes. Standard proteomics protocols fail to detect them as d/l substitutions do not affect the peptide parent and fragment masses. The differences in fragment yields are often limited, obstructing the investigations of important but low abundance epimers in isomeric mixtures. Separation of d/l-peptides using ion mobility spectrometry (IMS) was impeded by small collision cross section differences (commonly ∼1%). Here, broad baseline separation of DAACPs with up to ∼30 residues employing trapped IMS with resolving power up to ∼340, followed by time-of-flight mass spectrometry is demonstrated. The d/l-pairs coeluting in one charge state were resolved in another, and epimers merged as protonated species were resolved upon metalation, effectively turning the charge state and cationization mode into extra separation dimensions. Linear quantification down to 0.25% proved the utility of high resolution IMS-MS for real samples with large interisomeric dynamic range. Very close relative mobilities found for DAACP pairs using traveling-wave IMS (TWIMS) with different ion sources and faster IMS separations showed the transferability of results across IMS platforms. Fragmentation of epimers can enhance their identification and further improve detection and quantification limits, and we demonstrate the advantages of online mobility separated collision-induced dissociation (CID) followed by high resolution mass spectrometry (TIMS-CID-MS) for epimer analysis.

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

Lasso peptides are natural products characterized by a mechanically interlocked topology. The conformation of lasso peptides has been probed in the gas phase using ion mobility-mass spectrometry (IM-MS) which showed differences in the lasso and their unthreaded branched-cyclic topoisomers depending on the ion charge states. To further characterize the evolution of gas phase conformations as a function of the charge state and to assess associated changes in the hydrogen bond network, infrared multiple photon dissociation (IRMPD) action spectroscopy was carried out on two representative lasso peptides, microcin J25 (MccJ25) and capistruin, and their branched-cyclic topoisomers. For the branched-cyclic topoisomers, spectroscopic evidence of a disruption of neutral hydrogen bonds were found when comparing the 3+ and 4+ charge states. In contrast, for the lasso peptides, the IRMPD spectra were found to be similar for the two charge states, suggesting very little difference in gas phase conformations upon addition of a proton. The IRMPD data were thus found consistent and complementary to IM-MS, confirming the stable and compact structure of lasso peptides in the gas phase.

Rationale: Capistruin is a peptide synthesized by Burkholderia thailandensis E264, which displays a lasso topology. This knot-like structure confers interesting properties to peptides (e.g. antibacterial). Therefore, it is important to evaluate the sensitivity of structural characterization methods to such topological constraints., Methods: Ion mobility mass spectrometry (IMS-MS) experiments, using both drift tube and travelling wave instruments, were performed on lasso capistruin and on peptides with the same sequence, but displaying a branched-cyclic (un-threaded) or linear topology. Molecular dynamics (MD) simulations were then performed to further interpret the IMS results in terms of conformation., Results: The collision cross sections (CCSs) measured via IMS for the different forms of capistruin were found to be similar, despite their different topologies for the doubly charged species, but significant differences arise as the charge state is increased. MD simulations for the doubly charged linear peptide were consistent with the hypothesis that salt bridges are present in the gas phase. Moreover, through CCS measurements for peptides with site-specific mutations, the arginine residue at position 11 was found to play a major role in the stabilization of compact structures for the linear peptide., Conclusions: Differences in peptide topologies did not yield marked signatures in their respective IMS spectra. Such signatures were only visible for relatively high charge states, that allow Coulomb repulsion to force unfolding. At low charge states, the topologically unconstrained linear form of capistruin was found to adopt charge solvation-constrained structures, possibly including salt bridges, with CCSs comparable to those measured for the topologically constrained lasso form., (Copyright © 2015 John Wiley & Sons, Ltd.)

Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states, including the higher charge states obtained with sulfolane, yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the coexistence of fewer conformations and possible conformational changes.