Identification of single amino acid chiral and positional isomers using an electrostatically asymmetric nanopore

Abstract

Chirality is essential in nearly all biological organization and chemical reaction but is rarely considered due to technical limitations in identifying L/D isomerization. Using OmpF, a membrane channel from E. coli with an electrostatically asymmetric constriction zone, allows discriminating chiral amino acids in a single peptide. The heterogeneous distribution of charged residues in OmpF causes a strong asymmetric electrostatic field at the constriction. This asymmetry forces the sidechains of the peptides to specific orientations within OmpF, causing distinct ion current fluctuations. Using a statistical analysis of the respective ion current variations allows distinguishing the presence and position of each chiral amino acid. To explore potential applications, the disease-related peptide β-Amyloid, and its D-Asp1 isoform, as well as a mixture of the Icatibant peptide drug (HOE 140) and its D-Ser7 mutant, have been discriminated. Both chiral isomers were not applicable to be distinguished by mass spectrum approaches. These findings highlight a novel sensing mechanism for identifying single amino acids in single peptides and even for achieving single-molecule protein sequencing.

Nanopore-based technologies beyond DNA sequencing

“…This would provide a bottom-up approach for the production of customized chemicals. Presuming that a likely ultimate speed for the product formation is 1 ms per molecule, an array of 100 nanopores working in parallel would yield approximately 3.6 × 10 8 products in less than 1 h. Nanopores have also been increasingly used as force transducers, allowing the controlled localization, trapping and orientation of a diverse range of biomolecules for single-molecule biophysics studies 130,139 . Finally, nanopore-based biomedical applications have developed beyond DNA sequencing and epigenetic modification analyses, and are now used to sense molecular biomarkers (proteins, metabolites and nucleic acids) in biofluids and other biological specimens.…”