Publication in Chem. Sci.: Establishing Carbon-Carbon Double Bond Position and Configuration in Unsaturated Fatty Acids by Gas-Phase Infrared Spectroscopy
January 25, 2023


Fatty acids are an abundant class of lipids that are characterised by wide structural variation including isomeric diversity arising from the position and configuration of functional groups. Traditional approaches to fatty acid characterisation have combined chromatography and mass spectrometry for a description of the composition of individual fatty acids while infrared (IR) spectroscopy has provided insights into the functional groups and bond configurations at the bulk level. Here we exploit universal 3-pyridylcarbinol ester derivatization of fatty acids to acquire IR spectra of individual lipids as mass-selected gas-phase ions. Intramolecular interactions between the protonated pyridine moiety and carbon–carbon double bonds present highly sensitive probes for regiochemistry and configuration through promotion of strong and predictable shifts in IR resonances. Gas-phase IR spectra obtained from unsaturated fatty acids are shown to discriminate between isomers and enable the first unambiguous structural assignment of 6Z-octadecenoic acid in human-derived cell lines. Compatibility of 3-pyridylcarbinol ester derivatization with conventional chromatography-mass spectrometry and now gas-phase IR spectroscopy paves the way for comprehensive structure elucidation of fatty acids that is sensitive to regio- and stereochemical variations and with the potential to uncover new pathways in lipid metabolism.

C. Kirschbaum, R. S. E. Young, K. Greis, J. P. Menzel, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden, T. Causon, V. R. Narreddula, B. L. J. Poad, S. J. Blanksby, K. Pagel, Chem. Sci. (2023).




Publication in J. Am. Chem. Soc.: The Influence of the Electron Density in Acyl Protecting Groups on the Selectivity of Galactose Formation
October 26, 2022


The stereoselective formation of 1,2-cis-glycosidic bonds is a major bottleneck in the synthesis of carbohydrates. We here investigate how the electron density in acyl protecting groups influences the stereoselectivity by fine-tuning the efficiency of remote participation. Electron-rich C4-pivaloylated galactose building blocks show an unprecedented α-selectivity. The trifluoroacetylated counterpart with electron-withdrawing groups, on the other hand, exhibits a lower selectivity. Cryogenic infrared spectroscopy in helium nanodroplets and density functional theory calculations revealed the existence of dioxolenium-type intermediates for this reaction, which suggests that remote participation of the pivaloyl protecting group is the origin of the high α-selectivity of the pivaloylated building blocks. According to these findings, an α-selective galactose building block for glycosynthesis is developed based on rational considerations and is subsequently employed in automated glycan assembly exhibiting complete stereoselectivity. Based on the obtained selectivities in the glycosylation reactions and the results from infrared spectroscopy and density functional theory, we suggest a mechanism by which these reactions could proceed.

K. Greis, S. Leichnitz, C. Kirschbaum, C. W. Chang, M. H. Lin, G. Meijer, G. von Helden, P. H. Seeberger, K. Pagel, J. Am. Chem. Soc. (2022).




Publication in Eur. J. Org. Chem.: Neighboring Group Participation of Benzoyl Protecting Groups in C3- and C6-Fluorinated Glucose
March 25, 2022


Fluorination is a potent method to modulate chemical properties of glycans. Here, we study how C3- and C6-fluorination of glucosyl building blocks influence the structure of the intermediate of the glycosylation reaction, the glycosyl cation. Using a combination of gas-phase infrared spectroscopy and first-principles theory, glycosyl cations generated from fluorinated and non-fluorinated monosaccharides are structurally characterized. The results indicate that neighboring group participation of the C2-benzoyl protecting group is the dominant structural motif for all building blocks, correlating with the β-selectivity observed in glycosylation reactions. The infrared signatures indicate that participation of the benzoyl group in enhanced by resonance effects. Participation of remote acyl groups such as Fmoc or benzyl on the other hand is unfavored. The introduction of the less bulky fluorine leads to a change in the conformation of the ring pucker, whereas the structure of the active dioxolenium site remains unchanged.

K. Greis, C. Kirschbaum, G. Fittolani, E. Mucha, R. Chang, G. von Helden, G. Meijer, M. Delbianco, P. H. Seeberger, K. Pagel, Eur. J. Org. Chem. (2022).




Publication in Angew. Chem. Int. Ed.: Studying the Key Intermediate of RNA Autohydrolysis by Cryogenic Gas‐Phase Infrared Spectroscopy
March 1, 2022


Over the course of the COVID-19 pandemic, mRNA-based vaccines have gained tremendous importance. The development and analysis of modified RNA molecules benefit from advanced mass spectrometry and require sufficient understanding of fragmentation processes. Analogous to the degradation of RNA in solution by autohydrolysis, backbone cleavage of RNA strands was equally observed in the gas phase; however, the fragmentation mechanism remained elusive. In this work, autohydrolysis-like intermediates were generated from isolated RNA dinucleotides in the gas phase and investigated using cryogenic infrared spectroscopy in helium nanodroplets. Data from both experiment and density functional theory provide evidence for the formation of a five-membered cyclic phosphate intermediate and rule out linear or six-membered structures. Furthermore, the experiments show that another prominent condensed-phase reaction of RNA nucleotides can be induced in the gas phase: the tautomerization of cytosine. Both observed reactions are therefore highly universal and intrinsic properties of the investigated molecules.

K. Greis, C. Kirschbaum, M. I. Taccone, M. Götze, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden, K. Pagel, Angew. Chem. Int. Ed. (2022).




Publication in Anal. Bioanal. Chem.: Cryogenic infrared spectroscopy provides mechanistic insight into the fragmentation of phospholipid silver adducts
February 11, 2022


Tandem mass spectrometry is arguably the most important analytical tool for structure elucidation of lipids and other metabolites. By fragmenting intact lipid ions, valuable structural information such as the lipid class and fatty acyl composition are readily obtainable. The information content of a fragment spectrum can often be increased by the addition of metal cations. In particular, the use of silver ions is deeply rooted in the history of lipidomics due to their propensity to coordinate both electron-rich heteroatoms and C = C bonds in aliphatic chains. Not surprisingly, coordination of silver ions was found to enable the distinction of sn-isomers in glycerolipids by inducing reproducible intensity differences in the fragment spectra, which could, however, not be rationalized. Here, we investigate the fragmentation behaviors of silver-adducted sn- and double bond glycerophospholipid isomers by probing fragment structures using cryogenic gas-phase infrared (IR) spectroscopy. Our results confirm that neutral headgroup loss from silver-adducted glycerophospholipids leads to dioxolane-type fragments generated by intramolecular cyclization. By combining high-resolution IR spectroscopy and computational modelling of silver-adducted fragments, we offer qualitative explanations for different fragmentation behaviors of glycerophospholipid isomers. Overall, the results demonstrate that gas-phase IR spectroscopy of fragment ions can significantly contribute to our understanding of lipid dissociation mechanisms and the influence of coordinating cations.

C. Kirschbaum, K. Greis, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden, K. Pagel, Anal. Bioanal. Chem. (2022).
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Publication in Curr. Opin. Struct. Biol.: Gas-phase infrared spectroscopy of glycans and glycoconjugates
December 21, 2021


Glycans are intrinsically complex biomolecules that pose particular analytical challenges. Standard workflows for glycan analysis are based on mass spectrometry, often coupled with separation techniques such as liquid chromatography and ion mobility spectrometry. However, this approach does not yield direct structural information and cannot always distinguish between isomers. This gap might be filled in the future by gas-phase infrared spectroscopy, which has emerged as a promising structure-sensitive technique for glycan fingerprinting. This review highlights recent applications of gas-phase infrared spectroscopy for the analysis of synthetic and biological glycans and how they can be integrated into mass spectrometry-based workflows.

K. Greis, C. Kirschbaum, G. von Helden and K. Pagel, Curr. Opin. Struct. Biol., 72, 194-202 (2022).




Publication in JACS: Unveiling Glycerolipid Fragmentation by Cryogenic Infrared Spectroscopy
September 2, 2021


Mass spectrometry is routinely employed for structure elucidation of molecules. Structural information can be retrieved from intact molecular ions by fragmentation; however, the interpretation of fragment spectra is often hampered by poor understanding of the underlying dissociation mechanisms. For example, neutral headgroup loss from protonated glycerolipids has been postulated to proceed via an intramolecular ring closure but the mechanism and resulting ring size have never been experimentally confirmed. Here we use cryogenic gas-phase infrared (IR) spectroscopy in combination with computational chemistry to unravel the structures of fragment ions and thereby shed light on elusive dissociation mechanisms. Using the example of glycerolipid fragmentation, we study the formation of protonated five-membered dioxolane and six-membered dioxane rings and show that dioxolane rings are predominant throughout different glycerolipid classes and fragmentation channels. For comparison, pure dioxolane and dioxane ions were generated from tailor-made dehydroxyl derivatives inspired by natural 1,2- and 1,3-diacylglycerols and subsequently interrogated using IR spectroscopy. Furthermore, the cyclic structure of an intermediate fragment occurring in the phosphatidylcholine fragmentation pathway was spectroscopically confirmed. Overall, the results contribute substantially to the understanding of glycerolipid fragmentation and showcase the value of vibrational ion spectroscopy to mechanistically elucidate crucial fragmentation pathways in lipidomics.

C. Kirschbaum, K. Greis, L. Polewski, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden and K. Pagel, J. Am. Chem. Soc., 14314827-14834 (2021).



Publication in J. Phys. Chem. A: Chondroitin Sulfate Disaccharides in the Gas Phase: Differentiation and Conformational Constraints
May 12, 2021


Glycosaminoglycans (GAGs) are a family of complex carbohydrates vital to all mammalian organisms and involved in numerous biological processes. Chondroitin and dermatan sulfate, an important class of GAGs, are linear macromolecules consisting of disaccharide building blocks of N-acetylgalactosamine and two different uronic acids. The varying degree and the site of sulfation render their characterization challenging. Here, we combine mass spectrometry with cryogenic infrared spectroscopy in the wavenumber range from 1000 to 1800 cm–1. Fingerprint spectra were recorded for a comprehensive set of disaccharides bearing all known motifs of sulfation. In addition, state-of-the-art quantum chemical calculations were performed to aid the understanding of the differences in the experimental fingerprint spectra. The results show that the degree and position of charged sulfate groups define the size of the conformational landscape in the gas phase. The detailed understanding of cryogenic infrared spectroscopy for acidic and often highly sulfated glycans may pave the way to utilize the technique in fragment-based sequencing approaches.

M. Lettow, K. Greis, M. Grabarics, J. Horlebein, R. L. Miller, G. Meijer, G. von Helden and K. Pagel, J. Phys. Chem. A, 125, 4373-4379 (2021).



Publication in ABC: Non-covalent double bond sensors for gas-phase infrared spectroscopy of unsaturated fatty acids
May 6, 2021


The position and configuration of carbon-carbon double bonds in unsaturated fatty acids is crucial for their biological functions and influences health and disease. However, double bond isomers are not routinely distinguished by classical mass spectrometry workflows. Instead, they require sophisticated analytical approaches usually based on chemical derivatization and/or instrument modification. In this work, a novel strategy to investigate fatty acid double bond isomers (18:1) without prior chemical treatment or modification of the ion source was implemented by non-covalent adduct formation in the gas phase. Fatty acid adducts with sodium, pyridinium, trimethylammonium, dimethylammonium, and ammonium cations were characterized by a combination of cryogenic gas-phase infrared spectroscopy, ion mobility-mass spectrometry, and computational modeling. The results reveal subtle differences between double bond isomers and confirm three-dimensional geometries constrained by non-covalent ion-molecule interactions. Overall, this study on fatty acid adducts in the gas phase explores new avenues for the distinction of lipid double bond isomers and paves the way for further investigations of coordinating cations to increase resolution.


C. Kirschbaum, K. Greis, M. Lettow, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden and K. Pagel, Anal. Bioanal. Chem., 413, 3643-3653 (2021).



Publication in Nature Communications: Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy
February 22, 2021


Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.


C. Kirschbaum, K. Greis, E. Mucha, L. Kain, S. Deng, A. Zappe, S. Gewinner, W. Schöllkopf, G. von Helden, G. Meijer, P. B. Savage, M. Marianski, L. Teyton, K. Pagel, Nat. Commun., 12, 1201 (2021).



Publication in Organic Letters: Direct Experimental Characterization of the Ferrier Glycosyl Cation in the Gas Phase
November 5, 2020


The Ferrier rearrangement reaction is crucial for the synthesis of pharmaceuticals. Although its mechanism was described more than 50 years ago, the structure of the intermediate remains elusive. Two structures have been proposed for this Ferrier glycosyl cation: a 1,2-unsaturated cation that is resonance-stabilized within the pyranose ring or a cation that is stabilized by the anchimeric assistance of a neighboring acetyl group. Using a combination of gas-phase cryogenic infrared spectroscopy in helium nanodroplets and first-principles density functional theory, we provide the first direct structural characterization of Ferrier cations. The data show that both acetylated glucal and galactal lead to glycosyl cations of the dioxolenium type.


K. Greis, C. Kirschbaum, S. Leichnitz, S. Gewinner, W. Schöllkopf, G. von Helden, G. Meijer, P. H. Seeberger and K. Pagel, Org. Lett., 22, 8916-8919 (2020).



Publication in PCCP: Probing the conformational landscape and thermochemistry of DNA dinucleotide anions via helium nanodroplet infrared action spectroscopy
July 31, 2020


Isolation of biomolecules in vacuum facilitates characterization of the intramolecular interactions that determine three-dimensional structure, but experimental quantification of conformer thermochemistry remains challenging. Infrared spectroscopy of molecules trapped in helium nanodroplets is a promising methodology for the measurement of thermochemical parameters. When molecules are captured in a helium nanodroplet, the rate of cooling to an equilibrium temperature of ca. 0.4 K is generally faster than the rate of isomerization, resulting in “shock-freezing” that kinetically traps molecules in local conformational minima. This unique property enables the study of temperature-dependent conformational equilibria via infrared spectroscopy at 0.4 K, thereby avoiding the deleterious effects of spectral broadening at higher temperatures. Herein, we demonstrate the first application of this approach to ionic species by coupling electrospray ionization mass spectrometry (ESI–MS) with helium nanodroplet infrared action spectroscopy to probe the structure and thermochemistry of deprotonated DNA dinucleotides. Dinucleotide anions were generated by ESI, confined in an ion trap at temperatures between 90 and 350 K, and entrained in traversing helium nanodroplets. The infrared action spectra of the entrained ions show a strong dependence on pre-pickup ion temperature, consistent with the preservation of conformer population upon cooling to 0.4 K. Non-negative matrix factorization was utilized to identify component conformer infrared spectra and determine temperature-dependent conformer populations. Relative enthalpies and entropies of conformers were subsequently obtained from a van ’t Hoff analysis. IR spectra and conformer thermochemistry are compared to results from ion mobility spectrometry (IMS) and electronic structure methods. The implementation of ESI–MS as a source of dopant molecules expands the diversity of molecules accessible for thermochemical measurements, enabling the study of larger, non-volatile species.

D. A. Thomas, R. Chang, E. Mucha, M. Lettow, K. Greis, S. Gewinner, W. Schöllkopf, G. Meijer, and G. von Helden, Phys. Chem. Chem. Phys., 22, 18400-18413 (2020).


Publication in Analytical Chemistry: Cryogenic Infrared Spectroscopy Reveals Structural Modularity in the Vibrational Fingerprints of Heparan Sulfate Diastereomers
July 13, 2020


Heparan sulfate and heparin are highly acidic polysaccharides with a linear sequence, consisting of alternating glucosamine and hexuronic acid building blocks. The identity of hexuronic acid units shows a variability along their sequence, as D-glucuronic acid and its C5 epimer, L-iduronic acid, can both occur. The resulting backbone diversity represents a major challenge for an unambiguous structural assignment by mass spectrometry-based techniques. Here, we employ cryogenic infrared spectroscopy on mass-selected ions to overcome this challenge and distinguish isomeric heparan sulfate tetrasaccharides that differ only in the configuration of their hexuronic acid building blocks. High-resolution infrared spectra of a systematic set of synthetic heparan sulfate stereoisomers were recorded in the fingerprint region from 1000 to 1800 cm–1. The experiments reveal a characteristic combination of spectral features for each of the four diastereomers studied and imply structural modularity in the vibrational fingerprints. Strong spectrum-structure correlations were found and rationalized by state-of-the-art quantum chemical calculations. The findings demonstrate the potential of cryogenic infrared spectroscopy to extend the mass spectrometry-based toolkit for the sequencing of heparan sulfate and structurally related biomolecules.

M. Lettow, M. Grabarics, K. Greis, E. Mucha, M. Lettow, D. A. Thomas, P. Chopra, G.-J. Boons, R. Karlsson, J. E. Turnbull, G. Meijer, R. L. Miller, G. von Helden, and K. Pagel, Anal. Chem., 92, 10228-10232 (2020).


Publication in ChemPhysChem: The Impact of Leaving Group Anomericity on the Structure of Glycosyl Cations of Protected Galactosides
July 11, 2020


It has been reported that fragments produced by glycosidic bond breakage in mass spectrometry‐based experiments can retain a memory of their anomeric configuration, which has major implications for glycan sequencing. Herein, we use cryogenic vibrational spectroscopy and ion mobility‐mass spectrometry to study the structure of B‐type fragments of protected galactosides. Cationic fragments were generated from glycosyl donors carrying trichloroacetimidate or thioethyl leaving groups of different anomeric configuration. The obtained infrared signatures indicate that the investigated fragments exhibit an identical structure, which suggests that there is no anomeric memory in B‐type ions of fully protected monosaccharides.

K. Greis, E. Mucha, M. Lettow, D. A. Thomas, C. Kirschbaum, S. Moon, A. Pardo-Vargas, G. von Helden, G. Meijer, K. Gilmore, P. H. Seeberger, and K. Pagel, ChemPhysChem, 21, 1905-1907 (2020).


Publication in Angewandte Chemie: Resolving Sphingolipid Isomers using Cryogenic Infrared Spectroscopy
April 15, 2020


1‐Deoxysphingolipids are a recently described class of sphingolipids, which have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1‐deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon‐carbon double bond (C=C) isomers and hydroxylation‐regioisomers. Here we demonstrate that cryogenic gas‐phase infrared (IR) spectroscopy of ionized 1‐deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas‐phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome.

C. Kirschbaum, E. M. Saied, K. Greis, E. Mucha, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden, B. L. J. Poad, S. J. Blanksby, C. Arenz, and K. Pagel, Angew. Chem., Int. Ed., 59, 13638-13642 (2020).


Publication in Angewandte Chemie: Remote Participation during Glycosylation Reactions of Galactose Building Blocks: Direct Evidence from Cryogenic Vibrational Spectroscopy
January 16, 2020


The stereoselective formation of 1,2‐cis‐glycosidic bonds is challenging. However, 1,2‐cis‐selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short‐lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α‐selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium‐type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2‐cis‐glycosidic bonds.

M. Marianski, E. Mucha, K. Greis, S. Moon, A. Pardo, C. Kirschbaum, D. A. Thomas, G. Meijer, G. von Helden, K. Gilmore, P. H. Seeberger, and K. Pagel, Angew. Chem., Int. Ed., 59, 6166-6171 (2020).

Publication in Analytical & Bioanalytical Chemistry: IR action spectroscopy of glycosaminoglycan oligosaccharides
November 11, 2019


Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm−1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy.

M. Lettow, M. Grabarics, E. Mucha, D. A. Thomas, L. Polewski, J. Freyse, J. Rademann, G. Meijer, G. von Helden, and K. Pagel, Anal. Bioanal. Chem., 412, 533-537 (2020).


Publication in Angewandte Chemie: An Intrinsic Hydrophobicity Scale for Amino Acids and Its Application to Fluorinated Compounds
April 8, 2019


The classification of amino acids according to their intrinsic properties, such as the physico‐chemical properties and structure, yields fundamental insights into their role in interactions in biological processes. More than 100 hydrophobicity scales have been introduced, with each being based on a distinct condensed‐phase approach. However, a comparison of the hydrophobicity values gained from different techniques, and their relative ranking is not straightforward, as the interactions between the environment and amino acid are unique to each method. Here, we overcome this limitation by studying the properties of amino acids in the clean‐room environment of the gas phase. In the gas phase, entropic contributions from the hydrophobic effect are by default absent and only the side‐chain´s polarity dictates self‐assembly. This allows for the derivation of a novel hydrophobicity scale, which is solely based on the interaction between individual amino acid units within the cluster and thus more accurately reflects the intrinsic nature of a side‐chain. This principle can be further applied to classify non‐natural derivatives, as shown here for fluorinated amino acid variants.

W. Hoffmann, J. Langenhan, S. Huhmann, J. Moschner, R. Chang, M. Accorsi, J. Seo, R. Jörg, G. Meijer, B. Koksch, M. T. Bowers, G. von Helden, and K. Pagel, Angew. Chem., Int. Ed., 58, 8216-8220 (2019).

Publication in JACS: Characterization of a trans–trans Carbonic Acid–Fluoride Complex by Infrared Action Spectroscopy in Helium Nanodroplets
March 27, 2019


The high Lewis basicity and small ionic radius of fluoride promote the formation of strong ionic hydrogen bonds in the complexation of fluoride with protic molecules. Herein, we report that carbonic acid, a thermodynamically disfavored species that is challenging to investigate experimentally, forms a complex with fluoride in the gas phase. Intriguingly, this complex is highly stable and is observed in abundance upon nanoelectrospray ionization of an aqueous sodium fluoride solution in the presence of gas-phase carbon dioxide. We characterize the structure and properties of the carbonic acid–fluoride complex, F(H2CO3), and its deuterated isotopologue, F(D2CO3), by helium nanodroplet infrared action spectroscopy in the photon energy range of 390–2800 cm–1. The complex adopts a C2v symmetry structure with the carbonic acid in a planar trans–trans conformation and both OH groups forming ionic hydrogen bonds with the fluoride. Substantial vibrational anharmonic effects are observed in the infrared spectra, most notably a strong blue shift of the symmetric hydrogen stretching fundamental relative to predictions from the harmonic approximation or vibrational second-order perturbation theory. Ab initio thermostated ring-polymer molecular dynamics simulations indicate that this blue shift originates from strong coupling between the hydrogen stretching and bending vibrations, resulting in an effective weakening of the OH···F ionic hydrogen bonds.

D. A. Thomas, E. Mucha, M. Lettow, G. Meijer, M. Rossi, and G. von Helden, J. Am. Chem. Soc., 141, 5815-5823 (2019).

Publication in Analytical & Bioanalytical Chemistry: The role of the mobile proton in fucose migration
March 2, 2019

Fucose migration reactions represent a substantial challenge in the analysis of fucosylated glycan structures by mass spectrometry. In addition to the well-established observation of transposed fucose residues in glycan-dissociation product ions, recent experiments show that the rearrangement can also occur in intact glycan ions. These results suggest a low-energy barrier for migration of the fucose residue and broaden the relevance of fucose migration to include other types of mass spectrometry experiments, including ion mobility-mass spectrometry and ion spectroscopy. In this work, we utilize cold-ion infrared spectroscopy to provide further insight into glycan scrambling in intact glycan ions. Our results show that the mobility of the proton is a prerequisite for the migration reaction. For the prototypical fucosylated glycans Lewis x and blood group antigen H-2, the formation of adduct ions or the addition of functional groups with variable proton affinity yields significant differences in the infrared spectra. These changes correlate well with the promotion or inhibition of fucose migration through the presence or absence of a mobile proton.

M. Lettow, E. Mucha, C. Mainz, D. A. Thomas, M. Marianski, G. Meijer, G. von Helden, and K. Pagel, Anal. Bioanal. Chem., 411, 4637-4645 (2019).

Publication in Nature Communications: Unravelling the structure of glycosyl cations via cold-ion infrared spectroscopy
October 9, 2018

Glycosyl cations are the key intermediates during the glycosylation reaction that covalently links building blocks during the synthetic assembly of carbohydrates. The exact structure of these ions remained elusive due to their transient and short-lived nature. Structural insights into the intermediate would improve our understanding of the reaction mechanism of glycosidic bond formation. Here, we report an in-depth structural analysis of glycosyl cations using a combination of cold-ion infrared spectroscopy and first-principles theory. Participating C2 protective groups form indeed a covalent bond with the anomeric carbon that leads to C1-bridged acetoxonium-type structures. The resulting bicyclic structure strongly distorts the ring, which leads to a unique conformation for each individual monosaccharide. This gain in mechanistic understanding fundamentally impacts glycosynthesis and will allow to tailor building blocks and reaction conditions in the future.

E. Mucha, M. Marianski, F.-F. Xu, D. A. Thomas, G. Meijer, G. von Helden, P. H. Seeberger, and K. Pagel, Nat. Commun., 9, 4174 (2018).

Publication in The Journal of Physical Chemistry C: Structural Characterization of Molybdenum Oxide Nanoclusters Using Ion Mobility Spectrometry–Mass Spectrometry and Infrared Action Spectroscopy
September 7, 2018

Polyoxometalate clusters possess unique catalytic and electromagnetic properties. The structure and function of polyoxometalates is dictated by complex oligomerization processes, which in turn depend on the solution conditions. In this work, small gas-phase polyoxomolybdate nanoclusters (HMonO3n+11–, n = 1–8, and MonO3n+12–, n = 2–8) were investigated after nanoelectrospray of an acidified solution of ammonium heptamolybdate heptahydrate by ion mobility spectrometry–mass spectrometry (IMS–MS), infrared multiple photon dissociation (IRMPD) spectroscopy, and infrared action spectroscopy in helium nanodroplets. The spectra and collision cross sections obtained were matched to predictions from density-functional theory (DFT) to unravel the structural progression of nanoclusters with increasing size. For doubly charged clusters, transitions among chain (n = 2–3), ring (n = 4–5), and compact (n ≥ 6) structures are observed in IMS–MS and IR spectroscopy experiments, in agreement with low-energy structures from DFT calculations. For singly charged clusters, reduced Coulombic repulsion and hydrogen bonding interactions are found to strongly influence the most stable cluster structure. Notably, a noncovalent ring structure is observed for HMo3O101–, stabilized by a strong intramolecular hydrogen bond, and a compact structure is observed for HMo5O161–, in contrast to the ring structure favored for Mo5O162–.

M. Marianski, J. Seo, E. Mucha, D. A. Thomas, S. Jung, R. Schlögl, G. Meijer, A. Trunschke, and G. von Helden, J. Phys. Chem. C, 123, 7845-7853 (2019).

Publication in Angewandte Chemie: Ground‐State Structure of the Proton‐Bound Formate Dimer by Cold‐Ion Infrared Action Spectroscopy
June 19, 2018

The proton‐bound dicarboxylate motif, RCOO⋅H+OOCR, is a prevalent chemical configuration found in many condensed‐phase systems. The proton‐bound formate dimer HCOO⋅H+OOCH was studied utilizing cold‐ion IR action spectroscopy in the range 400–1800 cm−1. The spectrum obtained at ca. 0.4 K of ions captured in He nanodroplets was compared to that measured at ca. 10 K by photodissociation of Ar‐ion complexes. Similar band patterns are obtained by the two techniques that are consistent with calculations for a C2 symmetry structure with a proton shared equally between the two formate moieties. Isotopic substitution experiments point to the nominal parallel stretch of the bridging proton appearing as a sharp, dominant feature near 600 cm−1. Multidimensional anharmonic calculations reveal that the bridging proton motion is strongly coupled to the flanking −COO framework, an effect that is in line with the expected change in −C=O bond rehybridization upon protonation.

D. A. Thomas, M. Marianski, E. Mucha, G. Meijer, M. A. Johnson, and G. von Helden, Angew. Chem., Int. Ed., 57, 10615-10619 (2018).

Publication in Angewandte Chemie: Fucose Migration in Intact Protonated Glycan Ions - A Universal Phenomenon in Mass Spectrometry
April 24, 2018

Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision‐induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold‐ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low‐energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment.

E. Mucha, M. Lettow, M. Marianski, D. A. Thomas, W. B. Struwe, D. J. Harvey, G. Meijer, P. H. Seeberger, G. von Helden, and K. Pagel, Angew. Chem., Int. Ed., 57, 7440-7443 (2018).

Publication in Journal of Physical Chemistry Letters: Vibrational Spectroscopy of Fluoroformate, FCO2, Trapped in Helium Nanodroplets
April 18, 2018

Fluoroformate, also known as carbonofluoridate, is an intriguing molecule readily formed by the reductive derivatization of carbon dioxide. In spite of its well-known stability, a detailed structural characterization of the isolated anion has yet to be reported. Presented in this work is the vibrational spectrum of fluoroformate obtained by infrared action spectroscopy of ions trapped in helium nanodroplets, the first application of this technique to a molecular anion. The experimental method yields narrow spectral lines, providing experimental constraints on the structure that can be accurately reproduced using high-level ab initio methods. In addition, two notable Fermi resonances between a fundamental and combination band are observed. The electrostatic potential map of fluoroformate reveals substantial charge density on fluorine as well as on the oxygen atoms, suggesting multiple sites for interaction with hydrogen bond donors and electrophiles, which may in turn lead to intriguing solvation structures and reaction pathways.

D. A. Thomas, E. Mucha, S. Gewinner, W. Schöllkopf, G. Meijer, G. von Helden, J. Phys. Chem. Lett., 9 2305-2310 (2018).

Publication in JACS: The Structure of the Protonated Serine Octamer
April 16, 2018

The amino acid serine has long been known to form a protonated “magic-number” cluster containing eight monomer units that shows an unusually high abundance in mass spectra and has a remarkable homochiral preference. Despite many experimental and theoretical studies, there is no consensus on a Ser8H+ structure that is in agreement with all experimental observations. Here, we present the structure of Ser8H+ determined by a combination of infrared spectroscopy and ab initio molecular dynamics simulations. The three-dimensional structure that we determine is ∼25 kcal mol–1 more stable than the previous most stable published structure and explains both the homochiral preference and the experimentally observed facile replacement of two serine units.

V. Scutelnic, M. A. S. Perez, M. Marianski, S. Warnke, A. Gregor, U. Rothlisberger, M. T. Bowers, C. Baldauf, G. von Helden, T. R. Rizzo, J. Seo, J. Am. Chem. Soc., 140 7554-7560 (2018).

The article was highlighted in JACS spotlight as 'Serine Octamer: Possible Key to Biomolecule Handedness'


Publication in JACS: NFGAIL Amyloid Oligomers: The Onset of Beta-Sheet Formation and the Mechanism for Fibril Formation
December 14, 2017

The hexapeptide NFGAIL is a highly amyloidogenic peptide, derived from the human islet amyloid polypeptide (hIAPP). Recent investigations indicate that presumably soluble hIAPP oligomers are one of the cytotoxic species in type II diabe-tes. Here we use Thioflavin T staining, transmission electron microscopy as well as ion mobility-mass spectrometry coupled to infrared (IR) spectroscopy to study the amyloid formation mechanism and the quaternary- and secondary-structure of soluble NFGAIL oligomers. Our data reveal that at neutral pH NFGAIL follows a nucleation-dependent mechanism to form amyloid fibrils. During the lag phase, highly polydisperse, polymorph, and compact oligomers (oligomer number n=2-13) as well as extended intermediates (n=4-11) are present. IR secondary structural analysis reveals that compact conformations adopt turn-like structures, whereas extended oligomers exhibit a significant amount of β-sheet content. This agrees well with previous molecular dynamic simulations and provides direct experimental evidence that unordered off-pathway NFGAIL aggregates up to the size of at least the 13-mer as well as partially folded β-sheet containing oligomers are coexisting.

W. Hoffmann, K. Folmert, J. Moschner, X. Huang, H. von Berlepsch, B. Koksch, M. T. Bowers, G. von Helden, K. Pagel, J. Am. Chem. Soc., 140, 244–249 (2018).

Publication in Nature Chemistry: Infrared spectrum and structure of the homochiral serine octamer–dichloride complex
July 11, 2017

The amino acid serine is known to form a very stable octamer that has properties that set it apart from serine complexes of different sizes or from complexes composed of other amino acids. For example, both singly protonated serine octamers and anionic octamers complexed with two halogen ions strongly prefer homochirality, even when assembled from racemic D,L mixtures. Consequently, the structures of these complexes are of great interest, but no acceptable candidates have so far been identified. Here, we investigate anionic serine octamers coordinated with two chloride ions using a novel technique coupling ion mobility spectrometry–mass spectrometry with infrared spectroscopy, in combination with theoretical calculations. The results allow the identification of a unique structure for (Ser8Cl2)2− that is highly symmetric, very stable and homochiral and whose calculated properties match those observed in experiments.

J. Seo, S. Warnke, K. Pagel, M. T. Bowers, G. von Helden, Nat. Chem., 9, 1263–1268 (2017).

Publication in Angewandte Chemie: Glycan Fingerprinting via Cold-Ion Infrared Spectroscopy
June 12, 2017

The diversity of stereochemical isomers present in glycans and glycoconjugates poses a formidable challenge for comprehensive structural analysis. Typically, sophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However, coexisting structurally similar isomers often render an unambiguous identification impossible. Other powerful techniques such as gas-phase infrared (IR) spectroscopy have been limited to smaller glycans, since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit a wealth of well-resolved absorption features that are diagnostic for minute structural variations. The unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.

E. Mucha, A. I. González Flórez, M. Marianski, D. A. Thomas, W. Hoffmann, W. B. Struwe, H. S. Hahm, S. Gewinner, W. Schöllkopf, P. H. Seeberger, G. von Helden, K. Pagel, Angew. Chem. Int. Ed., 56, 11248–11251 (2017).

Publication in Current Opinion in Structural Biology: Ion mobility-mass spectrometry and orthogonal gas-phase techniques to study amyloid formation and inhibition
March 24, 2017

Amyloidogenic peptide oligomers are responsible for a variety of neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Due to their dynamic, polydisperse, and polymorphic nature, these oligomers are very challenging to characterize using traditional condensed-phase methods. In the last decade, ion mobility-mass spectrometry (IM-MS) and related gas-phase techniques have emerged as a powerful alternative to disentangle the structure and assembly characteristics of amyloid forming systems. This review highlights recent advances in which IM-MS was used to characterize amyloid oligomers and their underlying assembly pathway. In addition, we summarize recent studies in which IM-MS was used to size- and mass-select species for a further spectroscopic investigation and outline the potential of IM-MS as a tool for the screening of amyloid inhibitors.

W. Hoffmann, G. von Helden, and K. Pagel, Curr. Opin. Struct. Biol., 46, 7-15 (2017).

Publication in JACS: Stacking Geometries of Early Protoporphyrin IX Aggregates Revealed by Gas-Phase Infrared Spectroscopy
December 12, 2016

Amphiphilic porphyrins are of great interest in the field of supramolecular chemistry because they can be fabricated into highly ordered architectures that are stabilized by π–π stacking of porphine rings as well as by non-covalent interactions between their hydrophilic substituents. Protoporphyrin IX (PPIX) has two flexible propionic acid tails and is one of the most common amphiphilic porphyrins. However, unlike other PPIX analogues, PPIX does not form stable extended nanostructures, and the reason for this is still not understood. Here, we employ ion mobility mass spectrometry in combination with infrared multiple photon dissociation spectroscopy to investigate early aggregates of PPIX. The ion mobility results show that growth occurs via single-stranded face-to-face stacking of PPIX. From the infrared spectroscopy on well-defined aggregates, it can be concluded that pairing of the carboxylic acid groups of the tails is a stabilizing element and that such a pairing occurs across a third residue from residue n to residue n+2. The tetramer appears to be especially stable, because all of its propionic acid tails are optimally paired and no free tails to promote further growth are present, which possibly prevents PPIX from forming larger, well-ordered assemblies.

J. Seo, J. Jang, S. Warnke, S. Gewinner, W. Schöllkopf, and G. von Helden, J. Am. Chem. Soc., 138, 16315-16321 (2016).

Publication in JASMS: From Compact to String—The Role of Secondary and Tertiary Structure in Charge-Induced Unzipping of Gas-Phase Proteins
December 06, 2016

In the gas phase, protein ions can adopt a broad range of structures, which have been investigated extensively in the past using ion mobility-mass spectrometry (IM-MS)-based methods. Compact ions with low number of charges undergo a Coulomb-driven transition to partially folded species when the charge increases, and finally form extended structures with presumably little or no defined structure when the charge state is high. However, with respect to the secondary structure, IM-MS methods are essentially blind. Infrared (IR) spectroscopy, on the other hand, is sensitive to such structural details and there is increasing evidence that helices as well as β-sheet-like structures can exist in the gas phase, especially for ions in low charge states. Very recently, we showed that also the fully extended form of highly charged protein ions can adopt a distinct type of secondary structure that features a characteristic C5-type hydrogen bond pattern. Here we use a combination of IM-MS and IR spectroscopy to further investigate the influence of the initial, native conformation on the formation of these structures. Our results indicate that when intramolecular Coulomb-repulsion is large enough to overcome the stabilization energies of the genuine secondary structure, all proteins, regardless of their sequence or native conformation, form C5-type hydrogen bond structures. Furthermore, our results suggest that in highly charged proteins the positioning of charges along the sequence is only marginally influenced by the basicity of individual residues.

S. Warnke, W. Hoffmann, J. Seo, E. De Genst, G. von Helden and K. Pagel, J. Am. Soc. Mass Spectrom., 28, 638-646 (2017).

Publication in Nature Chemistry: An Infrared Spectroscopy Approach to Follow β-Sheet Formation in Peptide Amyloid Assemblies
September 26, 2016

Amyloidogenic peptides and proteins play a crucial role in a variety of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. These proteins undergo a spontaneous transition from a soluble, often partially folded form, into insoluble amyloid fibrils that are rich in β-sheets. Increasing evidence suggests that highly dynamic, polydisperse folding intermediates, which occur during fibril formation, are the toxic species in the amyloid-related diseases. Traditional condensed-phase methods are of limited use for characterizing these states because they typically only provide ensemble averages rather than information about individual oligomers. Here we report the first direct secondary-structure analysis of individual amyloid intermediates using a combination of ion mobility spectrometry–mass spectrometry and gas-phase infrared spectroscopy. Our data reveal that oligomers of the fibril-forming peptide segments VEALYL and YVEALL, which consist of 4–9 peptide strands, can contain a significant amount of β-sheet. In addition, our data show that the more-extended variants of each oligomer generally exhibit increased β-sheet content.

J. Seo, W. Hoffmann, S. Warnke, X. Huang, S. Gewinner, W. Schöllkopf, M. T. Bowers, G. von Helden and K. Pagel, Nat. Chem., 9, 39-44 (2017).

Publication in Angewandte Chemie: Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study
August 22, 2016

Can the structures of small to medium-sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion-mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β-lactoglobulin, which are prototypical examples of helical and β-sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.

J. Seo, W. Hoffmann, S. Warnke, M. T. Bowers, K. Pagel, and G. von Helden, Angew. Chem. Int. Ed., 55, 14173-14176 (2016). (German version)

Publication in PCCP: The impact of environment and resonance effects on the site of protonation of aminobenzoic acid derivatives
August 22, 2016

The charge distribution in a molecule is crucially determining its physical and chemical properties. Aminobenzoic acid derivatives are biologically active small molecules, which have two possible protonation sites: the amine (N-protonation) and the carbonyl oxygen (O-protonation). Here, we employ gas-phase infrared spectroscopy in combination with ion mobility-mass spectrometry and density functional theory calculations to unambiguously determine the preferred protonation sites of p-, m-, and o-isomers of aminobenzoic acids as well as their ethyl esters. The results show that the site of protonation does not only depend on the intrinsic molecular properties such as resonance effects, but also critically on the environment of the molecules. In an aqueous environment, N-protonation is expected to be lowest in energy for all species investigated here. In the gas phase, O-protonation can be preferred, and in those cases, both N- and O-protonated species are observed. To shed light on a possible proton migration pathway, the protonated molecule-solvent complex as well as proton-bound dimers are investigated.

J. Seo, S. Warnke, S. Gewinner, W. Schöllkopf, M. T. Bowers, K. Pagel, and G. von Helden, Phys. Chem. Chem. Phys., 18, 25474-25482 (2016).

Publication in Analyst: Gas-Phase Microsolvation of Ubiquitin: Investigation of Crown Ether Complexation Sites using Ion Mobility-Mass Spectrometry
July 27, 2016

In this study the gas-phase structure of ubiquitin and its lysine-to-arginine mutants was investigated using ion mobility-mass spectrometry (IM-MS) and electron transfer dissociation-mass spectrometry (ETD-MS). Crown ether molecules were attached to positively charged sites of the proteins and the resulting non-covalent complexes were analyzed. Collision induced dissociation (CID) experiments reveal relative energy differences between the wild type and the mutant crown-ether complexes. ETD-MS experiments were performed to identify the crown ether binding sites. Although not all of the binding sites could be revealed, the data confirm that the first crown ether is able to bind to the N-terminus. IM-MS experiments show a more compact structure for specific charge states of wild type ubiquitin when crown ethers are attached. However, data on ubiquitin mutants reveal that only specific lysine residues contribute to the effect of charge microsolvation. A compaction is only observed for one of the investigated mutants, in which the lysine has no proximate interaction partner. When the lysine residues are involved in salt bridges on the other hand, attachment of crown ethers has little effect on the structure.

M. Göth, F. Lermyte, X. J. Schmitt, S. Warnke, G. von Helden, F. Sobott, K. Pagel, Analyst, 141, 5465-5466 (2016).

Publication in PCCP: Assessing the Stability of Alanine-Based Helices by Conformer-Selective IR Spectroscopy
July 04, 2016

Polyalanine based peptides that carry a lysine at the C-terminus ([Ac-AlanLys + H]+) are known to form α-helices in the gas phase. Three factors contribute to the stability of these helices: ɪ) the interaction between the helix macro dipole and the charge, ɪɪ) the capping of dangling C=O groups by lysine and ɪɪɪ) the cooperative hydrogen bond network. In previous studies, the influence of the interaction between the helix dipole and the charge as well as the impact of the capping was studied intensively. Here, we complement these findings by systematically assessing the third parameter, the H-bond network. In order to selectively remove one H-bond along the backbone, we use amide-to-ester substitutions. The resulting depsi peptides were analyzed by ion-mobility and m/z-selective infrared spectroscopy as well as theoretical calculations. Our results indicate that peptides which contain only one ester bond still maintain the helical conformation. We conclude that the interaction between the charge and the helix macro-dipole is most crucial for the formation of the α-helical conformation and a single backbone H-bond has only little influence on the overall stability.

W. Hoffmann, M. Marianski, S. Warnke, J. Seo, C. Baldauf, G. von Helden, and K. Pagel, Phys. Chem. Chem. Phys., 18, 19950-19954 (2016).

Publication in Chemical Communications: Distinguishing N-Acetylneuraminic Acid Linkage Isomers on Glycopeptides by Ion Mobility-Mass Spectrometry
February, 2016

Differentiating the structure of isobaric glycopeptides represents a major challenge for mass spectrometry-based characterisation techniques. Here we show that the regiochemistry of the most common N-acetylneuraminic acid linkages of N-glycans can be identified in a site-specific manner from individual glycopeptides using ion mobility-mass spectrometry analysis of diagnostic fragment ions.

H. Hinneburg, J. Hofmann, W. B. Struwe, A. Thader, Friedrich Altmann, D. Varón Silva, P. H. Seeberger, K. Pagel, D. Kolarich, Chem. Commun., 52, 4381-4384 (2016).

Publication in Angewandte Chemie: Charge-induced unzipping of isolated proteins to a defined secondary structure
February, 2016

In our newest publication we present a combined experimental and theoretical study on the secondary structure of isolated proteins as a function of charge state. In infrared spectra of the proteins ubiquitin and cytochrome c, amide I (C=O stretch) and amide II (N–H bend) bands can be found at positions that are typical for condensed-phase proteins. For high charge states a new band appears, substantially red-shifted from the amide II band observed at lower charge states. The observations are interpreted in terms of Coulomb-driven transitions in secondary structures from mostly helical to extended C5-type hydrogen-bonded structures. Support for this interpretation comes from simple energy considerations as well as from quantum chemical calculations on model peptides. This transition in secondary structure is most likely universal for isolated proteins that occur in mass spectrometric experiments.

A. I. González Flórez, E. Mucha, D.-S. Ahn, S. Gewinner, W. Schöllkopf, K. Pagel, G. von Helden Angew. Chem. Int. Ed., 55 3295–3299 (2016). (German version)

Publication in Nature: Identification of carbohydrate anomers using ion mobility–mass spectrometry
September 30, 2015

Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes. However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides. Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers. Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers. Here, we demonstrate that ion mobility–mass spectrometry—a method that separates molecules according to their mass, charge, size, and shape—can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility–mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.

J. Hofmann, H. S. Hahm, S. Seeberger, K. Pagel Nature, 526, 241–244 (2015).

Publication: IR Spectroscopy of Protonated Leu-Enkephalin and its 18-crown-6 Complex Embedded in Helium Droplets
July 28, 2015

Ultracold IR spectra of the protonated five amino acid peptide leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) embedded in superfluid helium droplets have been recorded using a free-electron laser as radiation source. The results show resolved spectra, which are in good agreement with theoretical calculations, as well as with the available gas-phase data indicating that the helium environment does not induce a significant matrix-shift. In addition, the effect of the interaction between the charge and the peptide backbone has been further investigated by complexing protonated leu-enkephalin with one 18-crown-6 molecule. Good agreement between the experimental and theoretical results allow for an assignment of a preferred molecular structure.

A. I. González Flórez, D.-S. Ahn, S. Gewinner, W. Schöllkopf and G. von Helden, Phys. Chem. Chem. Phys., 17, 21902-21911 (2015).

Publication: Online Monitoring the Isomerization of an Azobenzene-Based Dendritic Bolaamphiphile Using Ion Mobility-Mass Spectrometry
April 20, 2015

In our newest publication ion mobility-mass spectrometry was used to obtain detailed information about the kinetics of the light-induced trans/cis isomerization process of a new supramolecular azobenzene-based bolaamphiphile. Further experiments revealed that the investigated light-induced structural transition dramatically influences the aggregation behaviour of the molecule.

L. Urner, B. Thota, O. Nachtigall, S. Warnke, G. von Helden, R. Haag, and K. Pagel, Chem. Commun., 51, 8801-8804 (2015).

Publication: Protomers of Benzocaine: Solvent and Permittivity Dependence
March 12, 2015

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid, which finds an application as a local anesthetic (LA), is found to adopt in its protonated form at least two populations of distinct structures in the gas phase and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization (ESI) process. Here we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry (IM-MS) to yield gas-phase IR spectra of simultaneously m/z and drift-time resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species - the N- and O-protonated form. Density functional theory (DFT) calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation has been suggested.

S. Warnke, J. Seo, J. Boschmans, F. Sobott, J.H. Scrivens, C. Bleiholder, M.T. Bowers, S. Gewinner, W. Schöllkopf, K. Pagel and G. von Helden, J. Am. Chem. Soc., 137, 4236–4242 (2015).

Invitation: Ion Mobility-Mass Spectrometry Workshop
February 24, 2015

We would like to cordially invite you and your co-workers to our workshop on ion mobility-mass spectrometry, which will take part from 26. to 27. March 2015 at the Fritz Haber Institute of the Max Planck Society in Berlin.
The workshop is especially aimed at PhD students and post docs working on IM-MS. At the first day there will be talks on the fundamentals and applications of IM-MS, while the second day will be devoted to practical work for which we will prepare four stations with hands-on experiments and modelling case studies.
To register, please write an email to with your affiliation and contact details until 20. March. The registration is free of charge.
More information can be found here.

We are looking forward to seeing you there.
Gert von Helden & Kevin Pagel

Publication: Exploring the conformational preferences of 20-residue peptides in isolation: Ac-Ala19-Lys + H+ vs. Ac-Lys-Ala19 + H+ and the current reach of DFT
February 24, 2015

Reliable, quantitative predictions of the structure of peptides based on their amino-acid sequence information are an ongoing challenge. We here explore the energy landscapes of two unsolvated 20-residue peptides that result from a shift of the position of one amino acid in otherwise the same sequence. Our main goal is to assess the performance of current state-of-the-art density-functional theory for predicting the structure of such large and complex systems, where weak interactions such as dispersion or hydrogen bonds play a crucial role. For validation of the theoretical results, we employ experimental gas-phase ion mobility-mass spectrometry and IR spectroscopy. While unsolvated Ac-Ala19-Lys + H+ will be shown to be a clear helix seeker, the structure space of Ac-Lys-Ala19 + H+ is more complicated. Our first-principles structure-screening strategy using the dispersion-corrected PBE functional (PBE + vdWTS) identifies six distinctly different structure types competing in the low-energy regime (≈16 kJ mol−1). For these structure types, we analyze the influence of the PBE and the hybrid PBE0 functional coupled with either a pairwise dispersion correction (PBE + vdWTS, PBE0 + vdWTS) or a many-body dispersion correction (PBE + MBD*, PBE0 + MBD*). We also take harmonic vibrational and rotational free energy into account. Including this, the PBE0 + MBD* functional predicts only one unique conformer to be present at 300 K. We show that this scenario is consistent with both experiments.

F. Schubert, M. Rossi, C. Baldauf, K. Pagel, S. Warnke, G. von Helden, F. Filsinger, P. Kupser, G. Meijer, M. Salwiczek, B. Koksch, M. Scheffler and V. Blum; Phys. Chem. Chem. Phys., 17, 7373-7385 (2015).

Publication: Analyzing the Higher Order Structure of Proteins with Conformer-selective Ultraviolet Photodissociation
February 2, 2015

The top-down approach in protein sequencing requires simple methods in which the analyte can be readily dissociated at every position along the backbone. In this context, ultraviolet photodissociation (UVPD) recently emerged as a promising tool because, in contrast to slow heating techniques such as collision induced dissociation (CID), the absorption of UV light is followed by a rather statistically distributed cleavage of backbone bonds. As a result, nearly complete sequence coverage can be obtained. It is well-known, however, that gas-phase proteins can adopt a variety of different, sometimes coexisting conformations and the influence of this structural diversity on the UVPD fragmentation behavior is not clear. Using ion mobility-UVPD-mass spectrometry we recently showed that UVPD is sensitive to the higher order structure of gas-phase proteins. In particular, the cis/trans isomerization of certain proline peptide bonds was shown to significantly influence the UVPD fragmentation pattern of two extended conformers of 11+ ubiquitin. Building on these results, we here provide conformer-selective UVPD data for 7+ ubiquitin ions, which are known to be present in a much more diverse and wider ensemble of different structures, ranging from very compact to highly extended species. Our data show that certain conformers fall into groups with similar UVPD fragmentation pattern. Surprisingly, however, the conformers within each group can differ tremendously in their collision cross section. This indicates that the multiple coexisting conformations typically observed for 7+ ubiquitin are caused by a few, not easily inter-convertible, subpopulations.

S. Warnke, G. von Helden, K. Pagel; Proteomics, 16, 2804–2812 (2015).

Publication: Native Like Helices in a Specially Designed β Peptide in the Gas Phase
January 12, 2015

In natural peptides, helices are stabilized by hydrogen bonds that point backward along the sequence direction. Until now, there is only little evidence for the existence of analogous structures in oligomers of conformationally unrestricted β amino acids. We specifically designed the β peptide Ac-(β2hAla)6-LysH+ to form native like helical structures in the gas phase. The design follows the known properties of the peptide Ac-Ala6-LysH+ that forms a α helix in isolation. We perform ion-mobility mass-spectrometry and vibrational spectroscopy in the gas phase, allied to state-of-the-art density-functional theory simulations of these molecular systems in order to characterize their structure. We can show that the straightforward exchange of alanine residues for the homologous β amino acids generates a system that is generally capable of adopting native like helices with backward oriented H-bonds. By pushing the limits of theory and experiments, we show that one cannot assign a single preferred structure type due to the densely populated energy landscape and present an interpretation of the data that suggests an equilibrium of three helical structures.

F. Schubert, K. Pagel, M. Rossi, S. Warnke, M. Salwiczek, B. Koksch, G. von Helden, V. Blum, C. Baldauf and M. Scheffler; Phys. Chem. Chem. Phys., 17, 5376-5385 (2015).

Publication: Estimating CCSs of Negatively Charged N-Glycans using TW IM-MS
October 1, 2014

In our most recent publication we demonstrate the calibration of travelling wave ion mobility-mass spectrometer to estimate CCS of carbohydrates. Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates is still challenging, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Ion mobility-mass spectrometry (IM-MS) is a promising technique for the separation and structural analysis of complex carbohydrate. Measured drift-times can be converted into collision cross sections (CCSs), which can be compared and implemented into databases. However, most of the currently used commercial IM-MS instruments utilize a non-uniform travelling wave field to propel the ions through the IM cell. As a result, CCSs measurements cannot be performed directly. Here, we present a calibration dataset for negatively charged N-glycans and their fragments. Moreover, we show that the well defined polysaccharide dextran is also a suitable calibrant for CCS estimations. In addition, our data indicate that a considerably increased error has to be taken into account when reference CCSs acquired in a different drift gas are used for calibration.

J. Hofmann, W. B. Struwe, C. A. Scarff, J. H. Scrivens, D. J. Harvey, K. Pagel, Anal. Chem., 86 (21), 10789–10795 (2014).

Publication: Photodissociation of Conformer-Selected Ubiquitin
June 30, 2014

In our most recent publication we investigated conformer-selected ubiquitin and their different ultraviolet photodissociation (UVPD) spectra.
In the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility–UVPD–mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11+ ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.

S. Warnke, C. Baldauf, M. Bowers, K. Pagel, G. von Helden; J. Am. Chem. Soc., 136 (29), 10308–10314 (2014).

The article also received a spotlight by Jeffrey M. Perkel.
J. Am. Chem. Soc., 136 (29), 10173–10173 (2014)

First FEL measurements!
December 13, 2013

At the beginning of December our group measured several IR spectra of biomolecules with the new FHI free electron laser (FEL). Stephan Warnke coupled his drift tube ion mobility-mass spectrometer to the FEL and recorded an IR spectrum of a mass- and conformer-selected Ubiquitin (see left picture).

Ana Isabel Gonzalez Florez and Doo-Sik Ahn observed the spectrum of Leucine Enkephalin (Tyr-Gly-Gly-Phe-Leu) in the wavelength range from 5 to 9 micron (see right picture). The ionized molecule was embedded in a superfluid He nano-droplet, which allowed the cooling to very low temperature (0.4 K). The ion was ejected from the droplet after photo excitation and enabled the measurement of narrow absorption lines. The spectra shown on the right correspond to the pure system (black) and to the peptide complexed with one crown ether (red).

Publication: How Cations Change Peptide Structure
July 12, 2013

Specific interactions between cations and proteins have a strong impact on peptide and protein structure. Herein, we shed light on the nature of the underlying interactions, especially regarding effects on the polyamide backbone structure. This was done by comparing the conformational ensembles of model peptides in isolation and in the presence of either Li+ or Na+, which can have different conformational effects on the same peptide, by using state-of-the-art density-functional theory (including van der Waals effects) and gas-phase infrared spectroscopy. We also assess the predictive power of current approximate density functionals for peptide-cation systems and compare to results with those of established protein force fields as well as high-level quantum chemistry calculations.

C. Baldauf, K. Pagel, S. Warnke, G. von Helden, B. Koksch, V. Blum, M. Scheffler; Chem. Eur. J. 19, 11224-11234 (2013).

Publication: Collision Cross Sections of Complex Carbohydrates for the Calibration of TW IM-MS Instruments
April 26, 2013

Due to their immense structural diversity and complexity it is still very challenging to fully characterize the structure of complex carbohydrates. A new and very promising technique to overcome these limitations is mobility-mass spectrometry (IM-MS). Here ions with identical atomic composition and mass, but different structure can be separated according to their shape and collision cross section (CCS). With the emergence of commercially available instruments in 2006 the technology became readily available. Because of the nonhomogeneous, travelling wave (TW) field utilized in these instruments, however, CCS values currently cannot be determined directly from the drift times measured. Instead, an external calibration using compounds of known CCS and similar molecular identity is required.
In this article we report an easy-to-follow calibration protocol for TW IM-MS instruments using a series of sodiated N-glycans, which were released from commercially available glycoproteins. Furthermore, our data clearly demonstrate that carbohydrate isomers with identical mass but different conformation can be distinguished based on their CCS.

K. Pagel, D. J. Harvey; Anal. Chem. 85, 5138-5145 (2013).

Publication: Side-Chain Microsolvation of Gas-Phase Proteins
January 15, 2013

There is ongoing debate on the extent to which protein structure is retained after transfer into the gas phase. Here, using ion mobility-mass spectrometry (IM-MS), we investigate the impact of side-chain backbone interactions on the structure of gas-phase protein ions by non-covalent attachment of crown ethers (CE). Our results indicate that in the absence of solvent, secondary interactions between charged lysine side chains and backbone carbonyls can significantly influence the structure of a protein. Once the charged residues are capped with CEs, certain charge states of the protein are found to undergo a significant structural compaction.

S. Warnke, G. von Helden, K. Pagel; J. Am. Chem. Soc. 135, 1177-1180 (2013).

The article was highlighted in Nature Chemistry as ‘Crowning Achievement’


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