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.

Misfolding and aggregation of α-Synuclein (α-Syn) play a central role in Parkinson’s disease (PD), with oligomeric intermediates implicated as key toxic species. Here, we investigate the aggregation of two α-Syn segments, the NACore (68GAVVTGVTAVA78, WT-PD1) and the preNAC region (47GVVHGVATVA56, WT-PD2), using high-resolution trapped ion-mobility mass spectrometry (TIMS-Qq-ToF) and Thioflavin T fluorescence spectroscopy. The NACore is the minimal sequence required for α-Syn aggregation, whereas most mutations affecting the onset of PD appear to be in the preNAC region, therefore modulating aggregation dynamics and toxicity. Our results demonstrate that TIMS-Qq-ToF effectively resolves and identifies oligomeric species, revealing distinct aggregation pathways for both peptide segments. Fluorescence assays confirm differences in aggregation kinetics and morphology, highlighting the different oligomer formation pathways observed with ion-mobility mass spectrometry. Despite the oligomer-preserving nature of the TIMS itself, ion transmission remains too harsh for the fragile oligomers, leading to fragmentation of fragile non-covalent assemblies. However, collisional cross-section (N2CCSTIMS) values support the presence of large oligomers. This study highlights the potential of TIMS-Qq-ToF for mapping α-Syn aggregation and underscores the need for optimised, soft ion transmission to better preserve fragile transient intermediates, ultimately contributing to a deeper understanding of PD.

The configuration of the first prototype timsOmni instrument, which integrates an Omnitrap linear ion trap into a timsTOF platform, is presented and applied to antibody analysis. A modified electrode design for the electron-based fragmentation (ExD) section of the Omnitrap platform is introduced, enhancing both the robustness and performance of the method. Optimal characterization of antibodies requires characterizing light and heavy chains as pairs in addition to sequencing their variable domains and identifying any modifications. This is best addressed using protein centric proteomics as heterogeneity information such as the specific clonal origin of each identified fragment can be retained. Furthermore, by acting on intact proteins that retain part of their structure such as disulfide bonds, it is possible to target selected regions for fragmentation such as some of the hypervariable complementarity determining regions (CDRs) that are unique for each clone and necessary for target recognition. Using electron-based fragmentation methods, we employed the prototype timsOmni mass spectrometer to obtain full CDR3 sequences for paired heavy and light chains with high confidence. Optimal results were obtained by performing Electron Induced Dissociation (EID) at ~35 eV electron energy, on native-like fragment antigen-binding (Fab) precursor ions. This approach yields both (a, x) as well as (c, z) fragment ion pairs with the potential to enhance both sequence coverage and annotation confidence. Overall, the timsOmni mass spectrometer presented here serves as an advanced and versatile platform for protein-centric proteomics, demonstrating exceptional sequencing power for comprehensive protein characterization.

Neurodegenerative diseases (ND), like Alzheimer’s and Parkinson’s, are age-related and are among the leading causes of death in the aging population. Although some medications alleviate symptoms, there is no cure. A common feature of ND is the presence of insoluble protein deposits found in the tissues of patients. The transition of proteins from a soluble and functional state to the formation of insoluble deposits, such as amyloid plaques composed of amyloid fibrils, is known as protein aggregation or self-assembly. ND-related proteins have aggregation cores, short peptide segments critical for aggregation, that can form amyloid fibrils and serve as model systems for method development. The peptide segments representing the aggregation cores of Aβ and tau proteins are the primary focus of this thesis. It describes the development and hyphenation of various analytical techniques, including infrared action spectroscopy, ion mobility mass spectrometry, and droplet-based microfluidics, for the study of the early stages of peptide aggregation. Chapter 1 reviews these techniques historically. Chapter 2 describes their principles, derived data, and limitations for peptide aggregation studies. Chapter 3 examines the structural properties of monomers and dimers of peptides with aromatic phenylalanine groups that form functional nanostructures using IR action spectroscopy. Chapter 4 focuses on the method development for improved peak assignment in ion mobility spectra of peptide segments within the aggregation core of tau protein. In Chapter 5, this ion mobility mass spectrometry (IM-MS) approach is used to study the structure and kinetics of tau peptide oligomers. By combining kinetic and structural data on amyloid fibrils obtained by bulk methods with insights into the properties of oligomers derived from IM-MS, a comprehensive understanding of the aggregation process can be achieved. Chapter 6 optimizes microfluidics and IM-MS for the study of peptide aggregation in droplets, addressing challenges, such as contaminant interference. Strategies to mitigate such problems are discussed. Finally, Chapter 7 summarizes the results and suggests future research directions.

The aggregation of the proteins tau and amyloid-β is a salient feature of Alzheimer’s disease, the most common form of neurodegenerative disorders. Upon aggregation, proteins transition from their soluble, monomeric, and functional state into insoluble, fibrillar deposits through a complex process involving a variety of intermediate species of differ-ent morphologies, including monomers, toxic oligomers, and insoluble fibrils. To control and direct peptide aggrega-tion, a complete characterization of all species present and an understanding of the molecular processes along the aggregation pathway are essential. However, this is extremely challenging due to the transient nature of oligomers and the complexity of the reaction networks. Therefore, we have employed a combined approach that allows us to probe the structure and kinetics of the emergence of oligomeric species, following them over time as they form fibrillar structures. Targeting the tau protein peptide segment Ac-PHF6-NH2, which is crucial for the aggregation of the full protein, soft nano-electrospray ionization combined with ion mobility mass spectrometry has been employed to study the kinetics of heparin-induced intact oligomer formation. The formed oligomers are identified and characterized using high-resolution ion mobility mass spectrometry, demonstrating that the addition of heparin does not alter the structure of the oligomeric species. The kinetics of fibril formation is monitored through a Thioflavin T fluorescence assay. Global fitting of the kinetic data indicates that secondary nucleation plays a key role in the aggregation of the Ac-PHF6-NH2 tau segment, while the primary nucleation rate is greatly accelerated by heparin.

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 the 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. Post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF (trapped ion mobility) spectrometer are introduced to improve oligomer assignment. In addition, TIMS collision cross section values are compared with travelling 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