Exploring the Aggregation Propensity of PHF6 Peptide Segments of the Tau Protein Using Ion Mobility Mass Spectrometry Techniques.

Peptide and protein aggregation involves the formation of oligomeric species, but the complex interplay between oligomers of different conformations and sizes complicates their structural elucidation. Using ion mobility mass spectrometry (IM-MS), we aim to reveal these early steps of aggregation for the Ac-PHF6-NH2 peptide segment from tau protein, thereby distinguishing between different oligomeric species and gaining an understanding of the aggregation pathway. An important factor that is often neglected, but which can alter the aggregation propensity of peptides, is the terminal capping groups. Here, we demonstrate the use of IM-MS to probe the early stages of aggregate formation of Ac-PHF6-NH2, Ac-PHF6, PHF6-NH2, and uncapped PHF6 peptide segments. The aggregation propensity of the four PHF6 segments is confirmed using thioflavin T fluorescence assays and transmission electron microscopy. A novel approach based on post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF (trapped ion mobility) spectrometer was developed to enhance oligomer assignment, especially for the higher-order aggregates. This approach pushes the limits of IM identification of isobaric species, whose signatures appear closer to each other with increasing oligomer size, and provides new insights into the interpretation of IM-MS data. In addition, TIMS collision cross section values are compared with traveling wave ion mobility (TWIMS) data to evaluate potential instrumental bias in the trapped ion mobility results. The two IM-MS instrumental platforms are based on different ion mobility principles and have different configurations, thereby providing us with valuable insight into the preservation of weakly bound biomolecular complexes such as peptide aggregates.

Gas-Phase Infrared Spectra of the C7H5 Radical and Its Bimolecular Reaction Products.

Resonance-stabilized radicals are considered as possible intermediates in the formation of polycyclic aromatic hydrocarbons (PAHs) in interstellar space. Here, we investigate the fulvenallenyl radical, the most stable C7H5 isomer by IR/UV ion dip spectroscopy employing free electron laser radiation in the mid-infrared region between 550 and 1750 cm-1. The radical is generated by pyrolysis from phthalide. Various jet-cooled reaction products are identified by their mass-selective IR spectra in the fingerprint region, based on a comparison with computed spectra. Interestingly, benzyl is present as a second resonance-stabilized radical. It is connected to fulvenallenyl by a sequence of two H atom losses or additions. Among the identified aromatic hydrocarbons are toluene and styrene, as well as polycyclic molecules, such as indene, naphthalene, fluorene and phenanthrene. Mechanisms for the formation of PAH from C7H5 have already been suggested in previous computational work. In particular, the radical/radical reaction of two fulvenallenyl radicals provides an efficient route to phenanthrene in one bimolecular step and might be relevant for PAH formation under astrochemical conditions.

Structural Properties of Phenylalanine-Based Dimers Revealed Using IR Action Spectroscopy.

Peptide segments with phenylalanine residues are commonly found in proteins that are related to neurodegenerative diseases. However, the self-assembly of phenylalanine-based peptides can be also functional. Peptides containing phenylalanine residues with different side caps, composition, and chemical alteration can form different types of nanostructures that find many applications in technology and medicine. Various studies have been performed in order to explain the remarkable stability of the resulting nanostructures. Here, we study the early stages of self-assembly of two phenylalanine derived peptides in the gas phase using IR action spectroscopy. Our focus lies on the identification of the key intra- and intermolecular interactions that govern the formation of the dimers. The far-IR region allowed us to distinguish between structural families and to assign the 2-(2-amino-2-phenylacetamido)-2-phenylacetic acid (PhgPhg) dimer to a very symmetric structure with two intermolecular hydrogen bonds and its aromatic rings folded away from the backbone. By comparison with the phenylalanine-based peptide cyclic L-phenylalanyl-L-phenylalanine (cyclo-FF), we found that the linear FF dimer likely adopts a less ordered structure. However, when one more phenylalanine residue is added (FFF), a more structurally organized dimer is formed with several intermolecular hydrogen bonds.

Probing the formation of isolated cyclo-FF peptide clusters by far-infrared action spectroscopy.

Small cyclic peptides containing phenylalanine residues are prone to aggregate in the gas phase into highly hydrophobic chains. A combination of laser desorption, mass spectrometry and conformational selective IR-UV action spectroscopy allows us to obtain detailed structural insights into the formation processes of the cyclic L-phenylalanyl-L-phenylalanine dipeptide (named cyclo-FF) aggregates. The rigid properties of cyclo-FF result in highly resolved IR spectra for the smaller clusters (n ≤ 3) and corresponding conformational assignments. For the higher order clusters (n > 3) the spectra are less resolved, however the observed ratios, peak positions and trends in IR shifts are key to make predictions on their structural details. Whereas the mid-IR spectral region between 1000-1800 cm-1 turns out to be undiagnostic for these small aggregates and the 3 µm region only for specific calculated structures, the far-IR contains valuable information that allows for clear assignments.

Do Xylylenes Isomerize in Pyrolysis?

We report infrared spectra of xylylene isomers in the gas phase, using free electron laser (FEL) radiation. All xylylenes were generated by flash pyrolysis. The IR spectra were obtained by monitoring the ion dip signal, using a IR/UV double resonance scheme. A gas phase IR spectrum of para-xylylene  was recorded, whereas ortho- and meta-xylylene were found to partially rearrange to benzocyclobutene and styrene. Computations of the UV oscillator strength  for all molecules were carried out and provde an explanation for the observation of the isomerization products.

Gas-Phase Infrared Spectroscopy of Neutral Peptides: Insights from the Far-IR and THz Domain.

Gas-phase, double resonance IR spectroscopy has proven to be an excellent approach to obtain structural information on peptides ranging from single amino acids to large peptides and peptide clusters. In this review, we discuss the state-of-the-art of infrared action spectroscopy of peptides in the far-IR and THz regime. An introduction to the field of far-IR spectroscopy is given, thereby highlighting the opportunities that are provided for gas-phase research on neutral peptides. Current experimental methods, including spectroscopic schemes, have been reviewed. Structural information from the experimental far-IR spectra can be obtained with the help of suitable theoretical approaches such as dynamical DFT techniques and the recently developed Graph Theory. The aim of this review is to underline how the synergy between far-IR spectroscopy and theory can provide an unprecedented picture of the structure of neutral biomolecules in the gas phase. The far-IR signatures of the discussed studies are summarized in a far-IR map, in order to gain insight into the origin of the far-IR localized and delocalized motions present in peptides and where they can be found in the electromagnetic spectrum.

The Gas-Phase Infrared Spectra of Xylyl Radicals.

The three isomers of the xylyl radical, C8H9, are possible intermediates in the formation of soot and polycyclic aromatic hydrocarbons (PAH). Their infrared spectra have been recorded by IR/UV ion dip spectroscopy using free electron laser radiation. The radicals were generated by flash pyrolysis from the corresponding nitrites and resonantly ionized via the D3 ← D0 transition around 310 nm. Mid-infrared spectra of the three xylyl isomers were recorded between 550 and 1700 cm-1 and are in excellent agreement with computations, provided that overtones and combination bands are included in the simulation. The results show that the three xylyl isomers can be distinguished by their infrared spectra and that no isomerization occurs in the pyrolysis reactor. The IR spectra obtained at m/z = 208 indicate that dimerization of xylyl radicals leads to substituted stilbenes, which has not been observed for benzyl.

Interactions of aggregating peptides probed by IR-UV action spectroscopy.

Peptide aggregation, the self-assembly of peptides into structured beta-sheet fibril structures, is driven by a combination of intra- and intermolecular interactions. Here, the interplay between intramolecular and formed inter-sheet hydrogen bonds and the effect of dispersion interactions on the formation of neutral, isolated, peptide dimers is studied using infrared action spectroscopy. Therefore, four different homo- and heterogenous dimers resulting from three different alanine-based model peptides have been formed under controlled and isolated conditions. The peptides differ from one another by the presence and location of a UV chromophore containing end cap. The conformations of the monomers of the peptides direct the final dimer structure: strongly bonded or folded structures result in weakly bound dimers. Here, intramolecular hydrogen bonds are favored over new intermolecular hydrogen bond interactions. In contrast, linear monomers are the ideal template to form parallel beta-sheet type structures. The weak intramolecular hydrogen bonds present in the linear monomers are replaced by the stronger inter-sheet hydrogen bond interactions. The influence of π-π dispersion interactions on the structure of the dimers is minimal, and the phenyl rings have a tendency to fold away from the peptide backbone to favour intermolecular hydrogen bond interactions over dispersion interactions. Quantum chemical calculations confirm our experimental observations.

Formation of Neutral Peptide Aggregates as Studied by Mass-Selective IR Action Spectroscopy.

The spontaneous aggregation of proteins and peptides is widely studied owing to its relation to neurodegenerative diseases. To understand the underlying principles of peptide aggregation, elucidation of structure and structural changes upon their formation is key. This level of detail can be obtained by studying the peptide self-assembly in the gas phase. Structural characterization of aggregates is mainly done on charged species, as adding charges is an intrinsic part of the technique to bring molecules into the gas phase. Studying neutral peptide aggregates will complement the existing picture. These studies are restricted to dimers due to experimental limitations. Herein, we present advances in laser desorption molecular beam spectroscopy to form neutral peptide aggregates consisting of up to 14 monomeric peptides in the gas phase. The combination of this technique with IR-UV spectroscopy allowed us to select each aggregate by size and subsequently characterize its structure.

Self-Reaction of ortho-Benzyne at High Temperatures Investigated by Infrared and Photoelectron Spectroscopy.

ortho-Benzyne, a Kekulé-type biradical is considered to be a key intermediate in the formation of polycyclic aromatic hydrocarbons (PAH) and soot. In the present work we study the ortho-benzyne self-reactions in a hot microreactor and identify the high-temperature products by IR/UV spectroscopy and by photoion mass-selected threshold photoelectron spectroscopy (ms-TPES) in a free jet. Ms-TPES confirms formation of ortho-benzyne as generated from benzocyclobutenedione, as well as benzene, biphenylene, diacetylene, and acetylene, originating from the reaction o-C6H4 → HCC-CCH + C2H2, and CH3. PAH molecules like naphthalene, 2-ethynylnaphthalene, fluorene, phenanthrene, and triphenylene are identified based on their IR/UV spectra. By comparison with recent computations their formation starting from o-benzyne can be readily understood and supports the importance of the biradical addition (1,4-cycloaddition followed by fragmentation) pathway to PAH molecules, recently proposed by Comandini et al.

Dimerization of the Benzyl Radical in a High-Temperature Pyrolysis Reactor Investigated by IR/UV Ion Dip Spectroscopy.

We investigate the self-reaction of benzyl, C7 H7 , in a high-temperature pyrolysis reactor. The work is motivated by the observation that resonance-stabilized benzyl radicals can accumulate in reactive environments and contribute to the formation of polycyclic aromatic hydrocarbons (PAHs) and soot. Reaction products are detected by IR/UV ion dip spectroscopy, using infrared radiation from the free electron laser FELIX, and are identified by comparison with computed spectra. Among the reaction products identified by their IR absorption are several PAHs linked to toluene combustion such as bibenzyl, phenanthrene, diphenylmethane, and fluorene. The identification of 9,10-dihydrophenanthrene provides evidence for a mechanism of phenanthrene formation from bibenzyl that proceeds by initial cyclization rather than an initial hydrogen loss to stilbene.