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.

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.