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.

IR/UV Double Resonance Study of the 2-Phenylallyl Radical and its Pyrolysis Products.

Isolated 2-phenylallyl radicals (2-PA), generated by pyrolysis from a nitrite precursor, have been investigated by IR/UV ion dip spectroscopy using free electron laser radiation. 2-PA is a resonance-stabilized radical that is considered to be involved in the formation of polycyclic aromatic hydrocarbons (PAH) in combustion, but also in interstellar space. The radical is identified based on its gas-phase IR spectrum. Furthermore, a number of bimolecular reaction products are identified, showing that the self-reaction as well as reactions with unimolecular decomposition products of 2-PA form several PAH efficiently. Possible mechanisms are discussed and the chemistry of 2-PA is compared with the one of the related 2-methylallyl and phenylpropargyl radicals.

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.

Developments in Trapped Ion Mobility Mass Spectrometry to Probe the Early Stages of Peptide Aggregation.

Ion mobility mass spectrometry (IM-MS) has proven to be an excellent method to characterize the structure of amyloidogenic protein and peptide aggregates, which are formed in coincidence with the development of neurodegenerative diseases. However, it remains a challenge to obtain detailed structural information on all conformational intermediates, originating from the early onset of those pathologies, due to their complex and heterogeneous environment. One way to enhance the insights and the identification of these early stage oligomers is by employing high resolution ion mobility mass spectrometry experiments. This would allow us to enhance the mobility separation and MS characterization. Trapped ion mobility spectrometry (TIMS) is an ion mobility technique known for its inherently high resolution and has successfully been applied to the analysis of protein conformations among others. To obtain conformational information on fragile peptide aggregates, the instrumental parameters of the TIMS-Quadrupole-Time-of-Flight mass spectrometer (TIMS-qToF-MS) have to be optimized to allow the study of intact aggregates and ensure their transmission toward the detector. Here, we investigate the suitability and application of TIMS to probe the aggregation process, targeting the well-characterized M307-N319 peptide segment of the TDP-43 protein, which is involved in the development of amyotrophic lateral sclerosis. By studying the influence of key parameters over the full mass spectrometer, such as source temperature, applied voltages or RFs among others, we demonstrate that by using an optimized instrumental method TIMS can be used to probe peptide aggregation.