Stephan Warnke

Protomers of Benzocaine: Solvent and Permittivity Dependence

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, 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 process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry 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 forms. Density functional theory 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 have been suggested.

Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds

Ultraviolet photodissociation (UVPD) of gas-phase proteins 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.

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

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.

An infrared spectroscopy approach to follow β-sheet formation in peptide amyloid assemblies

Amyloidogenic peptides and proteins play a crucial role in a variety of neurodegenerative disorders such as Alzheimers and Parkinsons 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.

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

Abstract 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.

Analyzing the higher order structure of proteins with conformer-selective ultraviolet photodissociation.

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 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‐MS, 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 interconvertible, subpopulations.

How Cations Change Peptide Structure

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)).

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

The charge distribution in a molecule is crucial in 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.

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

AbstractIn 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. Graphical Abstractᅟ

Stacking Geometries of Early Protoporphyrin IX Aggregates Revealed by Gas-Phase Infrared Spectroscopy

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.