Tiny Droplets, Large Insights: Studying Peptide Aggregation with Droplet-based Microfluidics Hyphenated with Ion Mobility Mass Spectrometry

Peptide and protein aggregation is a hallmark of numerous neurodegenerative diseases. However, the structural characterization of oligomeric intermediates remains challenging due to their low abundance, heterogenic nature, and the complexity of coinciding and transient reaction networks. Confining reactions to microenvironments, such as picoliter droplets in droplet-based microfluidics (DBMF), simplifies these networks and thereby enabling high-throughput analysis and controlled reaction kinetics. This study explores the integration of DBMF with ion mobility mass spectrometry (IM-MS) to probe aggregation kinetics in droplets. We address key technical challenges, including interference from the dispersed phase, surfactant, and DBMF device material, and we present optimization strategies for droplet generation, and surfactant concentration, to reduce contamination. Furthermore, we investigate instrumentation-based influences on ionization to improve signal stability. Using model peptides, bradykinin, leucine-enkephalin, and the aggregation-prone segment Ac-PHF6-NH₂ from the tau protein, we demonstrate the successful development of a sensitive method. Additionally, using the Ac-PHF6-NH₂ peptide, oligomeric transient species were probed and characterized by their IM and m/z values. This work demonstrates the potential of DBMF-IM-MS to study peptide aggregation in confined environments, providing insights into the formation of oligomeric species and laying the groundwork for studies on larger proteins and the development of therapeutic agents targeting aggregation using multi-channel devices.