Seeing the Invisibles: Detection of Peptide Enantiomers, Diastereomers, and Isobaric Ring Formation in Lanthipeptides Using Nanopores

Abstract

Mass spectrometry (MS) is widely used in proteomic analysis but cannot differentiate between molecules with the same mass-to-charge ratio. Nanopore technology might provide an alternative method for the rapid and cost-effective analysis and sequencing of proteins. In this study, we demonstrate that nanopore currents can distinguish between diastereomeric and enantiomeric differences in l- and d-peptides, not observed by conventional MS analysis, down to individual d-amino acids in small opioid peptides. Molecular dynamics simulations suggest that similar to chiral chromatography the resolution likely arises from multiple chiral interactions during peptide transport across the nanopore. Additionally, we used nanopore recordings to rapidly assess 4- and 11-amino acid ring formation in lanthipeptides, a process used in the synthesis of pharmaceutical peptides. The cyclization step requires distinguishing between constitutional isomers, which have identical MS signals and typically involve numerous tedious experiments to confirm. Hence, nanopore technology offers new possibilities for the rapid and cost-effective analysis of peptides, including those that cannot be easily differentiated by mass spectrometry.

Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning

“…Single-molecule electrical sensing using a nanopore is a promising method for biomarker peptide detection and discrimination. This has proven to be a potent tool in detecting and characterizing biological molecules, including nucleic acid sequencing and protein conformational analysis, − as well as defining peptide size, sequence variation, amino acid chirality, , aggregation, and post-translational modifications (PTMs) . Recently, this technology has been extended to the direct detection and characterization of biomarker molecules, proteins, and peptides, such as epidermal growth factor (EGF), angiotensin, and fibrinopeptide A. − In this work, we extend this method to detect the conformational variation adopted by two kinin biomarker peptides and use this information to discriminate between these peptides in a biofluid containing a complex mixture of molecules.…”

Silica Nanowires-Filled Glass Microporous Sensor for the Ultrasensitive Detection of Deoxyribonucleic Acid

“…Nanopore detection technology, as a novel single molecule detection method, can achieve molecular recognition by analyzing the ion current signals generated when molecules pass through nanopores with high sensitivity and specificity. − Solid-state nanopores have attracted much attention due to their controllable pore shape and size, excellent mechanical and chemical stability, and unique ion transport properties. − Due to the relatively excellent detection performance of solid-state nanopores, they have been widely used as a highly efficient detection device for ions, DNA, proteins, and so on. − Dekker et al first inserted α-hemolysin, which bound to DNA strands, into solid-state pores based on electrophoretic effects to form hybridized nanopores for the detection of single-stranded DNA . Seker et al used DNA probes labeled with nanoporous gold and methylene blue with different thicknesses and pore shapes for DNA target detection .…”