Niklas Geue

Native ion mobility−mass spectrometry (IM−MS) is a powerful biophysical technique for characterizing protein structure. Because individual charge states often exhibit distinct structural dynamics, accurate IM-MS measurements require charge states to be examined independently. Although anecdotal reports among mass spectrometrists suggest that protein ions can lose charges during native IM-MS experiments, a thorough investigation of this phenomenon has been lacking. Here, we demonstrate that protein ions undergo charge loss inside the mass spectrometer as a direct consequence of gas-phase collisions, potentially leading to altered charge state distributions and ion mobility data. This effect was consistently observed across two instrument platforms (Synapt G2-S and Q Exactive UHMR), both ion polarities (positive and negative mode) and for three proteins (bovine serum albumin, myoglobin, and immunoglobulin G). Systematic investigations of instrumental parameters revealed that collision gas pressure has the strongest impact on the magnitude of charge loss, and that higher charge states are more affected than lower charge states. We further show that this process is largely driven by the dissociation of salt adducts and can hence be minimized through desalting in solution or inside the mass spectrometer, prior to the first m/z-analyzer (usually a quadrupole). For collision-induced unfolding experiments, significant differences were found between fingerprints acquired with and without quadrupole selection, although it remains unclear whether this phenomenon is directly related to charge loss. Overall, our study provides fundamental insights into collision-induced charge loss as a source of error in native IM-MS measurements and discusses the relevance of quadrupole m/z-selection for improving confidence in IM studies.

Cryptophanes can selectively encapsulate small molecules and cations. Tuning host-guest selectivity requires insight into molecular recognition, which is analytically accessible with ion mobility mass spectrometry (IM-MS) via the conformational dynamics of complexation. Using activated IM-MS to differentiate between encapsulated and externally associated species, anti -cryptophanes complexes of the cation H ⁺ , Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , Mg ²⁺ , and Ag ⁺ were investigated. The m/z of fragments are characteristic of the binding mode of the host and guest pair. Using the combination of characteristic m/z and changes in ion mobility, IM-MS was sensitive to the host’s binding mode and provided further support to whether encapsulation had taken place. These findings demonstrate that activated IM-MS can sensitively probe cryptophane binding modes and provide new insight into the selectivity of cation encapsulation.

NMR, ion mobility mass spectrometry, gas-phase IR spectroscopy, molecular dynamics and DFT reveal why the reverse peptides GRGDS and SDGRG adopt distinct solution and gas-phase structures.

Native ion mobility–mass spectrometry (IM-MS) is a powerful biophysical technique for characterizing protein structure. Because individual charge states often exhibit distinct structural dynamics, accurate IM-MS measurements require charge states to be examined independently. Although anecdotal reports among mass spectrometrists suggest that protein ions can lose charges during native IM-MS experiments, a thorough investigation of this phenomenon has been lacking. Here, we demonstrate that protein ions undergo charge loss inside the mass spectrometer as a direct consequence of gas-phase collisions, potentially leading to altered charge state distributions and ion mobility data. This effect was consistently observed across two instrument platforms (Synapt G2-S and Q Exactive UHMR), both ion polarities (positive and negative mode) and for three proteins (bovine serum albumin, myoglobin, and immunoglobulin G). Systematic investigations of instrumental parameters revealed that collision gas pressure has the strongest impact on the magnitude of charge loss, and that higher charge states are more affected than lower charge states. We further show that this process is largely driven by the dissociation of salt adducts and can hence be minimized through desalting in solution or inside the mass spectrometer, prior to the first m/z -analyzer (usually a quadrupole). For collision-induced unfolding experiments, significant differences were found between fingerprints acquired with and without quadrupole selection, although it remains unclear whether this phenomenon is directly related to charge loss. Taken together, our study provides fundamental insights into collision-induced charge loss as a source of error in native IM-MS measurements and discusses the relevance of quadrupole m/z -selection for improving confidence in IM studies.

The Ferrier rearrangement of 1,2-unsaturated monosaccharides (glycals) to 2,3-unsatured glycosides, is important for the synthesis of pharmaceuticals and natural products. Mechanistically, the nucleophilic substitution proceeds via a cationic intermediate, the “Ferrier cation”, whose structure is difficult to capture with conventional analytical techniques. Here, we characterize the Ferrier cations of glucal and galactal building blocks involving aliphatic acyl protecting groups (pivaloyl-, levulinoyl- and chloroacetyl esters) using cryogenic gas-phase infrared spectroscopy. Comparing experimental and computed spectra of candidate structures, we established that all intermediates are stabilized by interactions of the C4 acyl protecting group with the C3 carbon of the pyranose ring (neighboring participation), potentially steering the stereochemical outcome of the Ferrier rearrangement. The resulting C4-C3-dioxolenium ion structure appears to be general to aliphatic acyl protecting groups, contributing to our fundamental understanding of Ferrier rearrangement reactions.

Constructing artificial assemblies that combine proteins and synthetic ligands has been hampered by the lack of protein–ligand interfaces that are sufficiently large and organized to enable precise structural control. Here ribosome display selection is used to identify a protein that binds a helical aromatic foldamer both tightly and selectively through a sizeable surface area. We used this complex as a supramolecular synthon to create well-defined hybrid foldamer–protein architectures. Examples include foldamers that bind two proteins and hold them at a precise distance, proteins that bind two foldamers and crystals in which proteins and foldamers are connected in cyclic or infinite arrays. The modularity of aromatic foldamers brings a further dimension to protein-based assemblies.

Controlling the stereoselective outcome of glycosidic bond formation remains a major challenge in chemical synthesis. This process typically proceeds along a complex SN1-SN2 mechanistic continuum. In solution, the key contributions to these mechanisms are difficult to determine due to influence by structural and environmental variables, such as solvents, counterions or temperature. To isolate the intrinsic factors driving steroselectivity in glycosylations, we investigate the reactions for the monosaccharide galactose in the gas phase. We perform ion–molecule galactosylation reactions in complete isolation inside an ion trap of a mass spectrometer and directly probe the stereochemistry of the formed products using mass-resolved ultraviolet cold ion spectroscopy. The results show that α-selectivity is significantly weaker in the gas phase than in solution, and that the product stereochemistry might be influenced by the nucleophile’s electron density rather than its overall nucleophilic strength.

Native ion mobility–mass spectrometry (IM-MS) is a powerful biophysical technique for characterizing protein structure. Because individual charge states often exhibit distinct structural dynamics, accurate IM-MS measurements require charge states to be examined independently. Although anecdotal reports among mass spectrometrists suggest that protein ions can lose charges during native IM-MS experiments, a thorough investigation of this phenomenon has been lacking. Here, we demonstrate that protein ions undergo charge loss inside the mass spectrometer as a direct consequence of gas-phase collisions, potentially leading to altered charge state distributions and ion mobility data. This effect was consistently observed across two instrument platforms (Synapt G2-S and Q Exactive UHMR), both ion polarities (positive and negative mode) and for three proteins (bovine serum albumin, myoglobin, and immunoglobulin G). Systematic investigations of instrumental parameters revealed that collision gas pressure has the strongest impact on the magnitude of charge loss, and that higher charge states are more affected than lower charge states. We further show that this process is largely driven by the dissociation of salt adducts and can hence be minimized through desalting in solution or inside the mass spectrometer, prior to the first m/z-analyzer (usually a quadrupole). For collision-induced unfolding experiments, no significant differences were found between fingerprints acquired with and without quadrupole selection, suggesting that the extent of charge loss is not sufficient to majorly interfere with such measurements. Taken together, our study provides fundamental insights into collision-induced charge loss as a source of error in native IM-MS measurements and discusses the relevance of quadrupole m/z-selection for improving confidence in IM studies.

Metallosupramolecular assemblies are promising platforms for selective extraction, catalysis, and molecular recognition, yet their solution-phase synthesis often relies on complex self-assembly pathways that can be difficult to control. Here, using ion mobility mass spectrometry supported by density functional theory calculations, we introduce a gas-phase approach for directing topology in polymetallic complexes based on the encapsulation of coordination metal cations within a homometallic {Cr₈} ring. Upon inclusion, the resulting host-guest complexes undergo collision-induced dissociation to produce distinct polymetallic fragments whose topologies, ring, host-guest complex or open chain, depend on the identity of the guest cation. This topology switching enables the targeted synthesis of new polymetallic rings, host-guest complexes and open chains in vacuo and provides insight into the stoichiometric and conformational preferences of such assemblies.

The reversed sequence pentapeptide pair GRGDS and SDGRG are widely used ion mobility spectrometry (IMS) standards as they exhibit distinct gas-phase mobilities despite identical mass and composition. Here, we investigate the molecular origins of their differing charge-state-dependent conformations using ion mobility mass spectrometry, cryogenic gas-phase infrared spectroscopy, NMR spectroscopy, molecular dynamics simulations, and density functional theory calculations. In aqueous solution, both peptides populate highly flexible conformational ensembles with only minor differences in compactness. Upon desolvation, however, the peptides collapse into distinct gas-phase structures stabilized by intramolecular hydrogen bonding. For the doubly protonated species, GRGDS adopts a compact cyclic “C-like” fold, whereas SDGRG forms a more extended “S-like” conformation driven by Coulombic repulsion between the protonated N-terminus and the arginine side chain. The singly charged ions show a more compact conformation for SDGRG compared to GRGDS, and a combination of different protomers and conformers appears to be present. Crown ether complexation and terminal glycine extensions of the doubly protonated species reduce the differences in IMS, supporting the importance of charge-charge interactions in defining the gas-phase folds. These findings provide molecular-level insight into a widely used IMS standard system and contribute to our fundamental understanding of structural rearrangements in peptides upon transfer to the gas phase.