MD Investigation on Separation and Purification of Peptides Utilizing Silicon Nitride Nanopores

Nanopore technology has emerged as a pivotal tool in biomolecular analysis owing to its high sensitivity, single-molecule resolution, and label-free detection capabilities. However, its application in peptide separation and purification has not been extensively explored. In this study, a novel peptide separation strategy using Si₃N₄ nanopores was proposed for the separation of three peptides─VEGF, FGF, and EGF─by integrating conventional molecular dynamics simulation, steered molecular dynamics, and umbrella sampling approach. Our simulation results demonstrate that the three peptides traverse Si₃N₄ nanopores with distinct energy barriers, indicating that the nanopores effectively differentiate between these similar peptides. This finding opens up possibilities for the separation of related peptides. Furthermore, we provide atomic-level insights into the underlying mechanisms responsible for the differences in translocation energy barriers among the peptides, thereby enriching the mechanistic understanding of peptide-nanopore interactions. Moreover, by analyzing the conformational dynamics of peptide translocation, we identify the crucial role of molecular sequence characteristics, such as length, polarity, hydrophobicity, and flexibility, in determining the peptide-nanopore interactions.