an asterisk indicates corresponding authorship
59 publications

Glycan analysis by ion mobility-mass spectrometry and gas-phase spectroscopy

Manz, C.; Pagel, K.*

Curr. Opin. Chem. Biol. 2018, 42, 16-24


Due to the existence of numerous isomers, the in-depth analysis of glycans represents a major challenge. Currently, the majority of glycans are analysed using mass spectrometry (MS)-based techniques, which can provide information on regioisomers but usually fail to differentiate stereoisomers. A promising approach to overcome this limitation is to implement ion mobility spectrometry (IMS) as an additional gas-phase separation dimension. This review highlights recent developments in which IM-MS was used as a tool for comprehensive glycan analysis or as rapid screening method for glycan feature analysis. Furthermore, we summarize a series of very recent investigations in which gas-phase spectroscopy is applied to study glycans and discuss the potential of the hyphenation between IM-MS and infrared (IR) spectroscopy as a future tool for glycomics and glycoproteomics.

Von normalen Proteinen zu unlöslichen Ablagerungen

Hoffmann, W.; Pagel, K.*

Nach. Chem. 2017, 65, 874–878

article (German)

Wenn Proteine sich falsch falten und aggregieren, also Amyloide bilden, entstehen neurodegenerative Erkrankungen wie Alzheimer oder Parkinson. Die toxischen Spezies sind kleine Oligomere, die sich aufgrund ihrer permanenten Strukturänderung nur schwer mit etablierten Methoden untersuchen lassen. Neuartige Gasphasenmethoden liefern vielversprechende erste Ergebnisse.

Structure and infrared spectrum of the homochiral serine octamer chloride adduct

Seo, J.*; Warnke, S.; Pagel, K.; Bowers, M. T.; von Helden, G.*

Nat. Chem. 2017, in press, doi: 10.1038/nchem.2821


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.

Side-chain effects on the structures of protonated amino acid dimers: A gas-phase infrared spectroscopy study

Seo, J.; Hoffmann, W.; Malerz, S.; Warnke, S.; Bowers, M. T.; Pagel, K.; von Helden, G.*

Int. J. Mass. Spectrom. 2017, in press, doi: 10.1016/j.ijms.2017.06.011


A protonated amino acid can interact in several ways with another uncharged amino acid molecule to form a protonated dimer. In case of amino acids that do not have basic or acidic side chains, the most likely protonation site is the amino group and the then protonated amine can be involved in a pairwise interaction with a neutral amine, a carboxylic acid, a carboxylate group and/or the sidechain of the partner amino acid. Here, we employ gas-phase infrared spectroscopy and density functional theory to identify these pairwise interactions in protonated homodimers of serine, isoleucine, phenylalanine and tyrosine. The results show the influence of the different side-chains on the respective interactions. A charge-solvated structure with pairwise interaction between a protonated amine and a neutral amine is preferred if the side chain can provide additional stabilizing interaction with the positive charge. In contrast, for amino acids where the side chain only interacts weakly with the protonated amine group, a protonated dimer is formed by an interaction between the protonated amine and the neutral carboxylic acid of the second amino acid.

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment-Based Screening

Göth, M.; Badock, V.; Weiske, J.; Pagel, K.; Kuropka, B.*

ChemMedChem 2017, 12, 1201–1211


Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention over the last years. So far, only a few studies discuss mass spectrometry as a screening technology for fragments. Here, we applied native electrospray ionization mass spectrometry (ESI-MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screen conducted by thermal shift assay (TSA) and represent different binding categories. Our data show that beside specific complex formation, many fragments show extensive multiple binding as well as charge-state shifts. Both of these factors complicate automated data analysis and lower the attractiveness of native MS as a primary screening tool for fragments. A comparison of hits identified by native MS and TSA shows good agreement for two proteins. Furthermore, we discuss general obstacles including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening.

Glycan Fingerprinting using Cold-Ion Infrared Spectroscopy

Mucha, E.; González Flórez, A. I; Marianski, M.; Thomas, D. A.; Hoffmann, W.; Struwe, W. B.; Hahm, H. S.; Gewinner, S.; Schöllkopf, W.; Seeberger, P. H.*; von Helden, G.*; Pagel K.*

Angew. Chem. Int. Ed. 2017, 56, 11248–11251

articlearticle (German Version)

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.

Ion mobility-mass spectrometry as a tool to investigate protein-ligand interactions

Göth, M.; Pagel, K.*

Anal. Bioanal. Chem. 2017, 409, 4305-4310


Ion mobility-mass spectrometry (IM-MS) is a powerful tool for the simultaneous analysis of mass, charge, size and shape of ionic species. It allows the characterization of even low-abundant species in complex samples and is therefore particularly suitable for the analysis of proteins and their assemblies. In the last years even complex and intractable species have been investigated successfully with IM-MS and the number of publications in this field is steadily growing. This trend article highlights recent advances in which IM-MS was used to study protein-ligand complexes and in particular focuses on the catch and release (CaR) strategy and collision induced unfolding (CIU).

Glycan Analysis by Ion Mobility-Mass Spectrometry

Hofmann, J. and Pagel, K.*

Ang. Chem. lnt. Ed. 2017, 56, 8342–8349

articlearticle (German Version)

Carbohydrates form one of the major classes of biological macromolecules in living organisms. To investigate their properties and function, an in-depth knowledge of their underlying structure is essential. However, the inherent structural complexity of glycans represents a major challenge. Carbohydrates are often branched and exhibit diverse regio- and stereochemistry. This in turn leads to a vast number of possible isomers, which are difficult to distinguish by using established analytical tools. In the last decade, ion mobility–mass spectrometry, a technique that separates ions based on their mass, charge, size, and shape, has emerged as a powerful alternative for isomer distinction. This Minireview highlights recent advances in ion mobility–mass spectrometry of complex carbohydrates and discusses its role in future analysis workflows.

Automated glycan assembly using the Glyconeer 2.1 synthesizer

Hahm, H. S.; Schlegel, M.; Hurevich, M.; Eller, S.; Schuhmacher, F.; Hofmann, J.; Pagel, K.; Seeberger, P. H.*

Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E3385-E3389


Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure–activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.

Ion mobility-mass spectrometry and orthogonal gas-phase techniques to study amyloid formation and inhibition

Hoffmann, W.; von Helden, G.; Pagel, K.*

Curr. Opin. Struct. Biol. 2017, 46, 7-15


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.

Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates

Huth, K.; Heek, T.; Achazi, K.; Kühne, C.; Urner, L. H.; Pagel, K.; Dernedde, J.*; Haag, R.*

Chem. Eur. J. 2017, 23, 4849-4862


A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels due to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels’ outstanding fluorescence quantum yields (FQYs) up to 100%. These coreunsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody Cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation via receptor-binding, cellular uptake and cytotoxicity studies revealed that labelling did not affect the antibody’s function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging.

Presynaptic calmodulin targets: Lessons from structural proteomics

Lipstein, N.; Göth, M.; Piotrowski, C.; Pagel, K.; Sinz, A.; Jahn, O.*

Exp. Rev. Prot. 2017, 14, 223-242


Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+ concentration into the regulation of synaptic transmission dynamics. We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes. Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM-targets for functional studies in vitro or ideally in vivo.

Identification of Lewis and Blood Group Carbohydrate Epitopes by Ion Mobility-Tandem-Mass Spectrometry Fingerprinting

Hofmann, J.; Stuckmann, A.; Crispin, M.; Harvey, D. J.; Pagel, K.* and Struwe, W. B.*

Anal. Chem. 2017, 89, 2318-2325


Glycans have several elements that contribute to their structural complexity, involving a range of monosaccharide building blocks, configuration of linkages between residues and various degrees of branching on a given structure. Their analysis remains challenging and resolving minor isomeric variants can be difficult, in particular terminal fucosylated Lewis and blood group antigens present on N- and O-glycans. Accurately characterizing these isomeric structures by current techniques is not straightforward and typically requires a combination of methods and/or sample derivatization. Yet the ability to monitor the occurrence of these epitopes is important as structural changes are associated with several human diseases. The use of ion mobility-mass spectrometry (IM-MS), which separates ions in the gas phase based on their size, charge and shape, offers a new potential tool for glycan analysis and recent reports have demonstrated its potential for glycomics. Here we show that Lewis and blood group isomers, which have identical fragmentation spectra, exhibit very distinctive IM drift times and collision cross sections (CCS). We show that IM-MS/MS analysis can rapidly and accurately differentiate epitopes from parotid N-glycans and milk oligosaccharides based on fucosylated fragment ions with characteristic CCSs.

Global N-glycan Site Occupancy of HIV-1 gp120 by High-Resolution Intact Mass Spectrometry

Struwe, W.*; Stuckmann, A.; Behrens, A.-J.; Pagel, K.; Crispin, M.*

ACS Chem. Biol. 2017, 12, 357-361


A vital step in HIV vaccine development strategies has been the observation that some infected individuals generate broadly neutralizing antibodies that target the glycans on the surface of HIV-1 gp120. These antibodies target glycan epitopes on viral envelope spikes and yet the positions and degree of occupancy of glycosylation sites is diverse. Therefore, there is a need to understand glycosylation occupancy on recombinant immunogens. The sheer number of potential glycosylation sites and degree of chemical heterogeneity impedes assessing the global sequon occupancy of gp120 glycoforms. Here, we trap the glycan processing of recombinant gp120 to generate homogenous glycoforms, facilitating occupancy assessment by intact mass spectrometry. We show that gp120 monomers of the BG505 strain contain either fully occupied sequenz or missing one and sometimes two glycans across the molecule. This biosynthetic engineering approach enables the analysis of therapeutically important glycoproteins otherwise recalcitrant to analysis by native mass spectrometry.

From Compact to String – The Role of Secondary and Tertiary Structure in Charge-Induced Unzipping of Gas-Phase Proteins


Warnke, S.; Hoffmann, W.; Seo, J.; De Genst, E.; von Helden, G.; Pagel, K.*

J. Am. Soc. Mass Spectrom. 2017, 28, 638-646


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.

An Infrared Spectroscopy Approach to Follow β-Sheet Formation in Peptide Amyloid Assemblies


Seo, J.; Hoffmann, W.; Warnke, S.; Huang, X.; Gewinner, S.; Schöllkopf, W.; Bowers, M. T.; von Helden, G.;* and Pagel, K.*

Nat. Chem. 2017, 9, 39-44


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.

Strukturen von Peptidaggregaten erforscht mit dem neuen Freie-Elektronen-Laser des Fritz-Haber-Instituts

von Helden, G.*; Schöllkopf, W.; Pagel, K.

Max Planck Gesellschaft - Jahrbuch 2016, doi: 10.17617/1.1I


Durch die Kombination von verschiedenen Trennmethoden konnten mithilfe des Freie-Elektronen-Lasers des Fritz-Haber-Instituts Infrarotspektren von größenselektierten Aggregaten von Peptidionen in der Gasphase gemessen werden. Die Spektren erlauben Rückschlüsse auf den Faltungszustand der Peptide, der von helikal zu β-Faltblatt variiert. Dieser Ansatz zur Faltungsbestimmung in Peptid- und Proteinaggregaten könnte zukünftig zum besseren Verständnis von Protein-Fehlfaltung und Aggregation und von den dadurch verursachten Krankheiten beitragen.

Ion Mobility Separation of Deprotonated Oligosaccharide Isomers - Evidence for Gas-phase Charge Migration

Struwe, W. B.*; Baldauf, C.*; Hofmann, J.; Rudd, P. M.; Pagel, K.*

Chem. Commun. 2016, 52, 12353-12356


There has been increasing evidence that certain isomeric glycans can be separated efficiently by ion mobility-mass spectrometry when deprotonated ions are analyzed. To better understand the fundamentals behind these separations, we here investigate the impact of ionisation mode and adduct formation using IM-MS, density-functional theory and ab initio molecular dynamics.

Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study


Seo, J.; Hoffmann, W.; Warnke, S.; Bowers, M. T.; Pagel, K.; von Helden, G.*

Angew. Chem. Int. Ed. 2016, 55, 14173-14176

articlearticle (German Version)

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.

The impact of environment and resonance effects on the site of protonation of aminobenzoic acid derivatives

Seo, J.; Warnke, S.; Gewinner, S.; Schöllkopf, W.; Bowers, M. T.; Pagel, K.*; von Helden, G.*

Phys. Chem. Chem. Phys. 2016, 18, 25474-25482


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.

Gas-Phase Microsolvation of Ubiquitin: Investigation of Crown Ether Complexation Sites using Ion Mobility-Mass Spectrometry


Göth, M.; Lermyte, F.; Schmitt, X. J.; Warnke, S.; von Helden, G.; Sobott, F.; Pagel, K.*

Analyst 2016, 141, 5502-5510


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 positive charge 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, these 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.

Travelling-wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N-glycans

Harvey, D. J.;* Scarff, C.; Edgeworth, M.; Pagel, K.; Thalassinos, K.; Struwe, W. B.; Crispin, M.; Scrivens, J.

J. Mass Spectrom. 2016, 51, 1064-1079


Nitrogen CCSs (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1-acid glycoprotein and thyroglobulin were measured with a travelling-wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man3GlcNAc3 from chicken ovalbumin and Man3GlcNAc3Fuc1 from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision-induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work and in the related earlier paper on high-mannose glycans, usually suggested that separations were due to conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated and it was found that complex glycans tended to have slightly smaller CCSs than high-mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility-m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs.

Conformational Shift of a β-Hairpin Peptide upon Complex Formation with an Oligo–proline Peptide Studied by Mass Spectrometry

Kölbel, K.; Warnke, S.; Seo, J.; von Helden, G.; Moretti, R.; Meiler, J.; Pagel, K.; Sinz, A.

ChemistrySelect 2016, 1, 3651-3656


So-called super-secondary structures such as the β-hairpin, studied here, form an intermediate hierarchy between secondary and tertiary structures of proteins. Their sequence-derived ‘pure’ peptide backbone conformation is combined with ‘remote’ interstrand or interresidue contacts reminiscent of the 3D-structure of full-length proteins. This renders them ideally suited for studying potential nucleation sites of protein folding reactions as well as intermolecular interactions. But β-hairpins do not merely serve as model systems; their unique structure characteristics warrant a central role in structural studies on their own. In this study we applied photo cross-linking in combination with high-resolution mass spectrometry and computational modeling as well as with ion mobility-mass spectrometry to elucidate these structural properties. Using variants of a known β-hairpin representative, the so-called trpzip peptide and its ligands, we found evidence for a conformational transition of the β-hairpin and its impact on ligand binding.

Assessing the Stability of Alanine-Based Helices by Conformer-Selective IR Spectroscopy

Hoffmann, W.; Marianski, M.; Warnke, S.; Seo, S.; Baldauf, C.; von Helden, G.; Pagel, K.*

Phys. Chem. Chem. Phys. 2016, 18, 19950-19954


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.

Identifizierung von isomeren Kohlenhydraten


Pagel, K.*; Hofmann, J.

GIT Laborfachzeitschrift 2016, 60, 32-34


Komplexe Oligosaccharide - oft auch Glycane genannt - sind Biomakromoleküle die eine essentielle Rolle bei der Proteinfaltung, der Zellerkennung, und dem Informationstransfer in lebenden Organismen spielen. Im Gegensatz zu linear verknüpften Biopolymeren wie DNA und Proteinen sind Glycane verzweigt und stereochemisch komplex, was eine enorme Herausforderung für ihre Charakterisierung darstellt. Die Struktur von Glycanen wird durch drei Parameter bestimmt: Komposition, Konnektivität und Konfiguration (Abb. 1). Die Komposition entspricht der Art der vorhandenen Monosaccharid-Bausteine. Diese Bausteine sind oft Isomere, besitzen also die gleiche Summenformel jedoch eine unterschiedliche Anordnung der Atome. Im Falle von Glucose (Glc) und Galactose (Gal) liegt der Unterschied zum Beispiel nur in der Stereochemie einer Hydroxylgruppe an einem bestimmten Kohlenstoffatom. Jedes Monosaccharid enthält wiederum mehrere Hydroxylgruppen die jeweils glycosidische Bindungen zum nächsten Baustein ausbilden können. Deshalb sind Glycane oft verzweigte Strukturen mit komplexer Regiochemie. Darüber hinaus können bei der Bildung einer glycosidische Bindung jeweils zwei Konfiguration entstehen, die sogenannte α- oder β-Konfiguration. Bereits zwei Bausteine können so in einer Vielzahl von Möglichkeiten miteinander verbunden werden und jedes Molekül besitzt dabei unterschiedliche chemische und physikalische Eigenschaften.

Photooxygenation and Gas-Phase Reactivity of Multiply Threaded Pseudorotaxanes

Nowosinski, K.; Warnke, S.; Pagel, K.; Komáromy, D.; Jiang, W.*; Schalley, C. A.*

J. Mass Spectrom. 2016, 51, 269-281


The solution-phase photooxygenation of multiply threaded crown/ammonium pseudorotaxanes containing anthracene spacers is monitored by electrospray ionization Fourier-transform ion-cyclotron-resonance (ESI-FTICR) mass spectrometry. The oxygenated pseudorotaxanes are mass-selected and fragmented by infrared multiphoton dissociation (IRMPD) and/or collision-induced dissociation (CID) experiments and and their behavior compared to that of the non-oxygenated precursors. [4+2]Cycloreversion reactions lead to the loss of O2, when no other reaction channel with competitive energy demand is available. Thus, the release of molecular oxygen can serve as a reference reaction for the energy demand of other fragmentation reactions such as the dissociation of the crown/ammonium binding motifs. The photooxygenation induces curvature into the initially planar anthracene and thus significantly changes the geometry of the divalent, anthracene-spacered wheel. This is reflected in ion-mobility data. Coulomb repulsion in multiply charged pseudorotaxanes assists the oxygen loss as the re-planarization of the anthracene increases the distance between the two charges.

Travelling-wave ion mobility and negative ion fragmentation of high mannose N-glycans

Harvey, D. J.; Scarff, C. A.; Edgeworth, M.; Struwe, W. B.; Pagel, K., Thalassinos, K.; Crispin, M.; Scrivens, J.

J. Mass Spectrom. 2016, 51, 219-235


The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed.

Distinguishing N-Acetylneuraminic Acid Linkage Isomers on Glycopeptides by Ion Mobility-Mass Spectrometry

Hinneburg, H.; Hofmann, J.; Struwe, W. B.; Thader, A.; Altmann, F.; Varón Silva, D.; Seeberger, P.; Pagel, K.*; Kolarich, D.*

Chem. Commun. 2016, 52, 4381-4384


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.

Charge-induced Unzipping of Isolated Proteins to a Defined Secondary Structure


González Flórez, A. I.; Mucha, E.; Ahn, D.-S.; Gewinner, S.; Schöllkopf, W.; Pagel, K.; von Helden, G.*

Angew. Chem. Int. Ed. 2016, 55, 3295-3299

articlearticle (German version)

Here 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.

Identification of Carbohydrate Anomers Using Ion Mobility-Mass Spectrometry


Hofmann, J.; Hahm, H. S.; Seeberger, P. H.*; Pagel, K.*

Nature 2015, 526, 241-244.


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.

GlycoMob: an Ion Mobility-Mass Spectrometry Collision Cross Section Database for Glycomics

Struwe, W.; Pagel, K.; Benesch, J.L.P.; Harvey, D.J.; Campbell, M.P.

Glycoconj. J. 2016, 33, 399-404


Ion mobility mass spectrometry (IM-MS) is a promising analytical technique for glycomics that separates glycan ions based on their collision cross section (CCS) and provides glycan precursor and fragment masses. It has been shown that isomeric oligosaccharide species can be separated by IM and identified on basis of their CCS and fragmentation. These results indicate that adding CCSs information for glycans and glycan fragments to searchable databases and analysis pipelines will increase identification confidence and accuracy. We have developed a freely accessible database, GlycoMob (http://​www.​glycomob.​org), containing over 900 CCSs values of glycans, oligosaccharide standards and their fragments that will be continually updated. We have measured the absolute CCSs of calibration standards, biologically derived and synthetic N-glycans ionized with various adducts in positive and negative mode or as protonated (positive ion) and deprotonated (negative ion) ions.

Collision cross sections of high-mannose N-glycans in commonly observed adduct states - Identification of gas-phase conformers unique to [M-H]- ions


Struwe, W. B. *; Benesch, J. L. P.; Harvey, D. J. and Pagel, K.*

Analyst 2015, 140, 6799-6803


We report collision cross sections (CCS) of high-mannose N-glycans as [M+Na]+, [M+K]+, [M+H]+, [M+Cl]-, [M+H2PO4]- and [M-H]- ions, measured by drift tube (DT) ion mobility-mass spectrometry (IM-MS) in helium and nitrogen gases. Further analysis using traveling wave (TW) IM-MS reveal the existence of distinct conformers exclusive to [M-H]- ions.

Online Monitoring the Isomerization of an Azobenzene-Based Dendritic Bolaamphiphile Using Ion Mobility-Mass Spectrometry


Urner, L.; Thota, B.; Nachtigall, O.; Warnke, S.; von Helden, G.; Haag, R.; Pagel, K.*

Chem. Commun. 2015, 51, 8801-8804


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.

Protomers of Benzocaine: Solvent and Permittivity Dependence


Warnke, S.; Seo, J.; Boschmans, J.; Sobott, F.; Scrivens, J. H.; Bleiholder, C.; Bowers, M. T.; Gewinner, S.; Schöllkopf, W.; Pagel, K.*; von Helden, G.*

J. Am. Chem. Soc. 2015, 137, 4236-4242


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.

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


Schubert, F.; Rossi, M.; Baldauf, C.; Pagel, K.; Warnke, S.; von Helden, G.; Filsinger, F.; Kupser, P.; Meijer, G.; Salwiczek, M.; Koksch, B.; Scheffler, M.; Blum, V.

Phys. Chem. Chem. Phys. 2015, 17, 7373-7385


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.

Analyzing the Higher Order Structure of Proteins with Conformer-Selective Ultraviolet Photodissociation


Warnke, S.; von Helden, G.; Pagel, K.*

Proteomics 2015, 16, 2804–2812


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.

Native like helices in a specially designed β peptide in the gas phase


Schubert, F.; Pagel, K.*; Rossi, M.; Warnke, S.; Salwiczek, M.; Koksch, B.; von Helden, G.; Blum, V.; Baldauf, C.; Scheffler, M.

Phys. Chem. Chem. Phys. 2015, 17, 5376-5385


In the 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.

Estimating Collision Cross Sections of Negatively Charged N-Glycans using Traveling Wave Ion Mobility-Mass Spectrometry

neg CCS estimation

Hofmann, J.; Struwe, W. B., Scarff, C. A.; Scrivens, J. H.; Harvey, D. J.; Pagel, K.*

Anal. Chem. 2014, 86, 10789-10795


Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates, however, is still one of the greatest challenges in modern glycoproteomics, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Recently, ion mobility-mass spectrometry (IM-MS), a technique in which ions are separated according to their mass, charge, and shape, has evolved as a promising technique for the separation and structural analysis of complex carbohydrates. This growing interest is based on the fact that the measured drift times can be converted into collision cross sections (CCSs), which can be compared, implemented into databases, and used as additional search criteria for structural identification. However, most of the currently used commercial IM-MS instruments utilize a nonuniform traveling wave field to propel the ions through the IM cell. As a result, CCS measurements cannot be performed directly and require calibration. Here, we present a calibration data set consisting of over 500 reference CCSs for negatively charged N-glycans and their fragments. Moreover, we show that dextran, already widely used as a calibrant in high performance liquid chromatography, is also a suitable calibrant for CCS estimations. Our data also 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.

Is there a Beta-Peptide Equivalent of the Alpha-Helix?

Baldauf, C.*; Schubert, F.; Pagel, K.; Warnke, S.; Rossi, M.; Salwiczek, M.; Koksch, B.; von Helden, G.; Blum, V.

Biophys. J. 2014, 106, 654a.


In organic chemistry, the concept of homologation describes the extension of a carbon chain by one methylene unit. The application of this concept to peptides gives rise to the class of β-peptides that feature an additional -CH2- unit in the monomer backbone. The study of such non-natural peptides offers insight into general folding mechanisms. The similarities to α-peptide structure combined with stability against proteases makes such foldamers promising scaffolds with possible applications in drug design.

Photodissociation of conformer-selected ubiquitin ions reveals site-specific cis/trans isomerization of proline peptide bonds


Warnke, S.; Baldauf, C.; Bowers, M.; Pagel, K.*; von Helden, G.

J. Am. Chem. Soc. 2014, 136 (29) , 10308–10314


Ultraviolet photodissociation of gas-phase proteins (UVPD) has attracted increased attention in recent years. This growing interest is largely based on the fact that – in contrast to slow heating techniques such as collision induced dissociation (CID) - the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, 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.

Energy-resolved ion mobility-mass spectrometry - A concept to improve the separation of isomeric carbohydrates


Hoffmann, W.; Hofmann, J.; Pagel, K.*

J. Am. Soc. Mass Spectrom. 2014, 25, 471-479


Recent works using ion mobility-mass spectrometry (IM-MS) have highlighted the power of this instrumental configuration to tackle one of the greatest challenges in glycomics and glycoproteomics: the existence of isobaric isomers. For a successful separation of species with identical mass but different structure via IM-MS, it is crucial to have sufficient IM resolution. In commercially available IM-MS instruments, however, this resolution is limited by the design of the instrument and usually cannot be increased at-will without extensive modifications. Here, we present a systematic approach to improve the resolving capability of IM-MS instruments using so-called energy-resolved ion mobility-mass spectrometry. The technique utilizes the fact that individual components in an isobaric mixture fragment at considerably different energies when activated in the gas phase via collision-induced dissociation (CID). As a result, certain components can be suppressed selectively at increased CID activation energy. Using a mixture of four isobaric carbohydrates, we show that each of the individual sugars can be resolved and unambiguously identified even when their drift times differ by as little as 3 %. However, the presented results also indicate that a certain difference in the gas-phase stability of the individual components is crucial for a successful separation via energy-resolved IM-MS.

Ion mobility-mass spectrometry of complex carbohydrates: Collision cross sections of sodiated N-linked glycans

Pagel, K.*; Harvey, D.J.

Anal. Chem. 2013, 85, 5138-5145


Currently, the vast majority of complex carbohydrates are characterized using mass spectrometry (MS)-based techniques. Measuring the molecular mass of a sugar, however, immediately poses a fundamental problem: entire classes of the constituting monosaccharide building blocks exhibit an identical atomic composition and, consequently, also an identical mass. Therefore, carbohydrate MS data can be highly ambiguous and often it is simply not possible to clearly assign a particular molecular structure. A promising approach to overcome the above-mentioned limitation is to implement an additional gas-phase separation dimension using ion mobility spectrometry (IMS), which is a method in which molecules of identical mass and structure but different structure can be separated according to their shape and collision cross section (CCS). With the emergence of commercially available hybrid ion mobility–mass spectrometry (IM-MS) instruments in 2006, IMS technology became readily available. Because of the nonhomogeneous, traveling 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. Here, we report a calibration protocol for TW IMS instruments using a series of sodiated N-glycans that were released from commercially available glycoproteins using an easy-to-follow protocol. The underlying CCS values were determined using a modified Synapt HDMS instrument with a linear drift tube, which was described in detail previously. Our data indicate that, under in-source fragmentation conditions, only a few glycans are required to obtain a TW IMS calibration of sufficient quality. In this context, however, the type of glycan was shown to be of tremendous importance. Furthermore, our data clearly demonstrate that carbohydrate isomers with identical mass but different conformation can be distinguished based on their CCS when all the associated errors are taken into account.

How cations change peptide structure

Baldauf, C.; Pagel, K.*; Warnke, S.; von Helden, G.; Koksch, B.; Blum, V.; Scheffler, M.

Chem. Eur. J. 2013, 19, 11224-11234


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+ by using state-of-the-art density-functional theory (including van der Waals effects) and gas-phase infrared spectroscopy. These monovalent cations have a drastic effect on the local backbone conformation of turn-forming peptides, by disruption of the hydrogen-bonding networks, thus resulting in severe distortion of the backbone conformations. In fact, Li+ and Na+ can even have different conformational effects on the same peptide. 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 (CCSD(T)).

Protein structure in the gas phase: The influence of side-chain microsolvation

Warnke, S.; von Helden, G.; Pagel, K.*

J. Am. Chem. Soc. 2013, 135, 1177-1180


There is ongoing debate about the extent to which protein structure is retained after transfer into the gas phase. Here, using ion-mobility spectrometry, we investigated the impact of side-chain–backbone interactions on the structure of gas-phase protein ions by noncovalent attachment of crown ethers (CEs). 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 significant structural compaction.

Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase

Pagel, K.; Natan, E.; Hall, Z.; Fersht, A.R.; Robinson, C.V.

Angew. Chem. Intl. Ed. 2013, 52, 361-365


Spontaneous shrinking: The intrinsically disordered tumor suppressor protein p53 was analyzed by using a combination of ion mobility mass spectrometry and molecular dynamics simulations. Structured p53 subdomains retain their overall topology upon transfer into the gas phase. When intrinsically disordered segments are introduced into the protein sequence, however, the complex spontaneously collapses in the gas phase to a compact conformation.

Local Conformational Preferences of Peptides Near Attached Cations: Structure Determination by First-Principles Theory and IR-Spectroscopy

Baldauf, C.*; Pagel, K.; Warnke, S.; von Helden, G.; Meijer, G.; Koksch, B.; Blum, V.; Scheffler, M.

Biophys. J. 2012, 102, 46a.


In the transition from secondary to tertiary structure in peptides and proteins, turns take a special role. They are the hinges that arrange the periodic secondary structure elements (helices and strands) to the native fold. It is a known effect that Li+ alters peptide backbone structure, and we investigate this effect on the structure and dynamics of turns for model peptides Ac-Ala-Ala-Pro-Ala-NMe (AAPA) and Ac-Ala-Asp-Pro-Ala-NMe (ADPA) by theoretical conformational predictions and experimental vibrational spectroscopy.

Structure analysis of an amyloid-forming model peptide by a systematic glycine and proline scan

Gerling, U.I.M.; Brandenburg, E.; Berlepsch, H.V.; Pagel, K.; Koksch, B.

Biomacromolecules 2011, 12, 2988-2996


The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.

Interaction of the p53 DNA-binding domain with its N-terminal extension modulates the stability of the p53 tetramer

Natan, E.; Baloglu, C.; Pagel, K.; Freund, S.M.V.; Morgner, N.; Robinson, C.V.; Fersht, A.R.; Joerger, A.C.

J. Mol. Biol. 2011, 409, 358-368


The tetrameric tumor suppressor p53 plays a pivotal role in the control of the cell cycle and provides a paradigm for an emerging class of oligomeric, multidomain proteins with structured and intrinsically disordered regions. Many of its biophysical and functional properties have been extrapolated from truncated variants, yet the exact structural and functional role of certain segments of the protein is unclear. We found from NMR and X-ray crystallography that the DNA-binding domain (DBD) of human p53, usually defined as residues 94–292, extends beyond these domain boundaries. Trp91, in the hinge region between the disordered proline-rich N-terminal domain and the DBD, folds back onto the latter and has a cation–π interaction with Arg174. These additional interactions increase the melting temperature of the DBD by up to 2 °C and inhibit aggregation of the p53 tetramer. They also modulate the dissociation of the p53 tetramer. The absence of the Trp91/Arg174 packing presumably allows nonnative DBD–DBD interactions that both nucleate aggregation and stabilize the interface. These data have important implications for studies of multidomain proteins in general, highlighting the fact that weak ordered–disordered domain interactions can modulate the properties of proteins of complex structure.

Secondary structure of Ac-Alan-LysH+ polyalanine peptides (n = 5,10,15) in vacuo: Helical or not?

Rossi, M.; Blum, V.; Kupser, P.; von Helden, G.; Bierau, F.; Pagel, K.; Meijer, G.; Scheffler, M.

J. Phys. Chem. Lett. 2010, 1, 3465-3470


The polyalanine-based peptide series Ac-Alan-LysH+ (n = 5−20) is a prime example that a secondary structure motif that is well-known from the solution phase (here: helices) can be formed in vacuo. Here we revisit the series members n = 5,10,15, using density functional theory (van der Waals corrected generalized gradient approximation) for structure predictions, which are then corroborated by room temperature gas-phase infrared vibrational spectroscopy. We employ a quantitative comparison based on Pendry’s reliability factor (popular in surface crystallography). In particular, including anharmonic effects into calculated spectra by way of ab initio molecular dynamics produces remarkably good experiment−theory agreement. We find the longer molecules (n = 10,15) to be firmly α-helical in character. For n = 5, calculated free-energy differences show different H-bond networks to still compete closely. Vibrational spectroscopy indicates a predominance of α-helical motifs at 300 K, but the lowest-energy conformer is not a simple helix.

Alternate dissociation pathways identified in charge-reduced protein complex ions

Pagel, K.; Hyung, S.-J.; Ruotolo, B.T.; Robinson, C.V.

Anal. Chem. 2010, 82, 5363-5372


Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

Amide-I and -II vibrations of the cyclic β-sheet model peptide gramicidin S in the gas phase

Kupser, P.; Pagel, K., Oomens, J., Polfer, N., Koksch, B., Meijer, G., von Helden, G.

J. Am. Chem. Soc. 2010, 132, 2085-2093


In the condensed phase, the peptide gramicidin S is often considered as a model system for a β-sheet structure. Here, we investigate gramicidin S free of any influences of the environment by measuring the mid-IR spectra of doubly protonated (deuterated) gramicidin S in the gas phase. In the amide I (i.e., C═O stretch) region, the spectra show a broad split peak between 1580 and 1720 cm−1. To deduce structural information, the conformational space has been searched using molecular dynamics methods and several structural candidates have been further investigated at the density functional level. The calculations show the importance of the interactions of the charged side-chains with the backbone, which is responsible for the lower frequency part of the amide I peak. When this interaction is inhibited via complexation with two 18-crown-6 molecules, the amide I peak narrows and shows two maxima at 1653 and 1680 cm−1. A comparison to calculations shows that for this complexed ion, four C═O groups are in an antiparallel β-sheet arrangement. Surprisingly, an analysis of the calculated spectra shows that these β-sheet C═O groups give rise to the vibrations near 1680 cm−1. This is in sharp contrast to expectations based on values for the condensed phase, where resonances of β-sheet sections are thought to occur near 1630 cm−1. The difference between those values might be caused by interactions with the environment, as the condensed phase value is mostly deduced for β-sheet sections that are embedded in larger proteins, that interact strongly with solvent or that are part of partially aggregated species.

Gas-phase IR spectra of intact α-helical coiled coil protein complexes

Pagel, K.*; Kupser, P.; Bierau, F.; Polfer, N.C.; Steill, J.D.; Oomens, J.; Meijer, G.; Koksch, B.; von Helden, G.

Int. J. Mass Spectrom. 2009, 283, 161-168


Electrospray ionization (ESI) is the softest ionization method that is currently available and it is widely accepted, that ESI generated ions of proteins and protein assemblies at certain conditions retain characteristic aspects of their solution-state conformation. ESI mass spectrometry (MS) therefore evolved as a useful tool to obtain information on composition, stoichiometry, and dynamics of non-covalently associated protein complexes. While tertiary structure information of proteins can be obtained from ion mobility spectrometry (IMS), only a few techniques yield direct information on the secondary structure of gas-phase peptides and proteins. We present here the mid-IR spectroscopic secondary structural analysis of three de novo designed α-helical coiled coil model peptides and their non-covalently associated complexes in the gas-phase. The conformational stability of such coiled coil peptides in solution is primarily driven by aggregation. Isolated monomers usually remain unfolded. Two of the investigated peptides were designed to assemble into stable α-helical complexes in acidic solution, while the third one remains monomeric and unfolded at these conditions. Monomer ions of all three peptides show comparable photodissociation IR spectra and therefore suggest an unfolded conformation in the gas phase. In contrast, considerable C=O stretch (amide-I) and N–H bend (amide-II) band shifts have been observed for the dimers which is consistent with an elevated H-bond content. These findings provide evidence that at least a fraction of the condensed phase α-helical structure is retained in the gas-phase coiled coil complexes.

Following polypeptide folding and assembly with conformational switches


Pagel, K.; Koksch, B.

Curr. Opin. Chem. Biol. 2008, 12, 730-739


Conformational transitions are a crucial factor in the vast majority of protein misfolding diseases. In most of these cases, the change in conformation is accompanied by the formation of insoluble aggregates, which often precludes a detailed characterization at the molecular level. Therefore, much effort has been put into the development of simplified, easy-to-synthesize peptide models that can be used to elucidate the molecular processes that underlie the conformational switch. For a design to be successful, two, sometimes concomitantly fulfilled, requirements are of importance. First, it is essential to create inherent structural ambiguity. This is usually achieved by combining the most prominent characteristics of different folds within a consensus sequence. Second, a stimulus-sensitive functionality that responds to alterations in the environment, such as pH, ionic strength or the presence of metal ions, is often needed to control structural conversion and to shift the equilibrium in either direction.

Intramolecular charge interactions as a tool to control the coiled-coil-to-amyloid transformation

Pagel, K.; Wagner, S.C.; Araghi, R.R.; von Berlepsch, H.; B”ttcher, C.; Koksch, B.

Chem. Eur. J. 2008, 14, 11442-11451


Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insoluble β-sheet rich fibrils. Molecular-level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the β-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH-controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α-helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid-like fibrils. Peptide B features a glutamate-based switch functionality and exhibits opposite pH-dependent folding behavior. In this case, α-helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.

How metal ions affect amyloid formulation: Cu2+- and Zn2+-sensitive peptides

Pagel, K.; Seri, T.; von Berlepsch, H.; Griebel, J.; Kirmse, R.; B”ttcher, C.; Koksch, B.

ChemBioChem 2008, 9, 531-536


The common feature of proteins involved in many neurodegenerative diseases is their ability to adopt at least two different stable conformations. The conformational transition that shifts the equilibrium from the functional, mostly partially α-helical structure, to the β-sheet rich amyloid can be triggered by numerous factors, such as mutations in the primary structure or changes in the environment. We present a set of model peptides that, without changes in their primary structure, react in a predictable fashion in the presence of transition metal ions by adopting different conformations and aggregate morphologies. These de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled-coil structural motif. Furthermore, domains that favor β-sheet formation have been incorporated to make the system prone to amyloid formation. As a third feature, histidine residues create sensitivity towards the presence of transition metal ions. CD spectroscopy, ThT fluorescence experiments, and transmission electron microscopy were used to characterize peptide conformation and aggregate morphology in the presence of Cu2+ and Zn2+. Furthermore, the binding geometry within peptide–Cu2+ complexes was characterized by electron paramagnetic resonance spectroscopy.

Random coils, β-sheet ribbons, and α-helical fibers: One peptide adopting three different secondary structures at will

Pagel, K.; Wagner, S.C.; Samedov, K.; von Berlepsch, H.; Böttcher, C.; Koksch, B.

J. Am. Chem. Soc. 2006, 128, 2196-2197


To potentially cure neurodegenerative diseases, we need to understand on a molecular level what triggers the complex folding mechanisms and shifts the equilibrium from functional to pathological isoforms of proteins. The development of small peptide models that can serve as tools for such studies is of paramount importance. We describe the de novo design and characterization of an α-helical coiled coil based model peptide that contains structural elements of both α-helical folding and β-sheet formation. Three distinct secondary structures can be induced at will by adjustment of pH or concentration. Low concentrations at pH 4.0 yield globular particles of the unfolded peptide, while at the same pH, but at higher concentration, defined β-sheet ribbons are formed. In contrast, at high concentrations and pH 7.4, the peptide forms highly ordered α-helical fibers. Thus, this system allows one to systematically study now the consequences of the interplay between peptide and protein primary structure and environmental factors for peptide and protein folding on a molecular level.

Directing the secondary structure of polypeptides at will: From helices to amyloids and back again?

Pagel, K.; Vagt, T.; Koksch, B.

Org. Biomol. Chem. 2005, 3, 3843-3850


An ageing society faces an increasing number of neurodegenerative diseases such as Alzheimer's, Parkinson’s, and Creutzfeld–Jacob disease. The deposition of amyloid fibrils is a pathogenic factor causing the destruction of neuronal tissue. Amyloid-forming proteins are mainly α-helical in their native conformation, but undergo an α-helix to β-strand conversion before or during fibril formation. Partially unfolded or misfolded β-sheet fragments are discussed as direct precursors of amyloids. To potentially cure neurodegenerative diseases we need to understand the complex folding mechanisms that shift the equilibrium from the functional to the pathological isoform of the proteins involved. This paper describes a novel approach that allows us to study the interplay between peptide primary structure and environmental conditions for peptide and protein folding in its whole complexity on a molecular level. This de novo designed peptide system may achieve selective inhibition of fibril formation.

From α-helix to β-sheet - A reversible metal ion induced peptide secondary structure switch

Pagel, K.; Vagt, T.; Kohajda, T.; Koksch, B.

Org. Biomol. Chem. 2005, 3, 2500-2502


Here we introduce a peptide model based on an α-helical coiled coil peptide, providing a simple system which can be used for a systematic study of the impact of different metal ions in different oxidation states on peptide secondary structure on a molecular level; histidine residues were incorporated into the heptad repeat to generate possible complexation sites for Cu2+ and Zn2+ ions.

Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide


Pagel, K.; Seeger, K.; Seiwert, B.; Villa, A.; Mark, A.E.; Berger, S.; Koksch, B.

Org. Biomol. Chem. 2005, 3, 1189-1194


We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modification of two hydrophobic core positions by Phe/[15N,13C]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept. This finding is also supported by molecular dynamics simulations. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in combination with NMR experiments was used for verification of the oligomerization equilibria of the coiled coil peptide.