Attempts to Understand and Manipulate the Orientation and Retention of Peptides Inside Nanopores Expand the Toolbox for Nanopore-Based Proteomics

Nanopore techniques have emerged as promising tools for deciphering complex proteomics from the single-molecule level by profiling individual peptides. The entry orientation of a peptide and its subsequent retention inside the nanopore are crucial for the analytical results but remain challenging to be properly defined, which limits the sensing capability of nanopore-based proteomics technology. Here, we propose that electrically driving peptide entry into α-hemolysin nanopores against opposing electroosmotic flows is a promising platform to guide the polypeptide with a preferential orientation. α-Hemolysin mutants with opposite ion selectivity have been engineered and employed to measure peptides at pH 3.8 and at alkaline pH exceeding 10.0. The impact of peptide charge distribution on the orientation has been investigated by simulating the peptide dipole moment and electrophysiology measurements. In addition, the retention position of the peptide has been altered by introducing tryptophan residues at position 125 within the β-barrel region of α-hemolysin, which increases the sensitivity for specific peptide region and enables the detection of initially inaccessible monomethylation modifications. Thus, we present a rational framework to investigate and manipulate the orientation and dwelling position of peptides inside the α-hemolysin nanopore, which can enhance our capability to profile polypeptides by using nanopore techniques.