Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore.

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

Biological nanopores for single-molecule sensing.

Evolution has found countless ways to transport material across cells and cellular compartments separated by membranes. Protein assemblies are the cornerstone for the formation of channels and pores that enable this regulated passage of molecules in and out of cells, contributing to maintaining most of the fundamental processes that sustain living organisms. As in several other occasions, we have borrowed from the natural properties of these biological systems to push technology forward and have been able to hijack these nano-scale proteinaceous pores to learn about the physical and chemical features of molecules passing through them. Today, a large repertoire of biological pores is exploited as molecular sensors for characterizing biomolecules that are relevant for the advancement of life sciences and application to medicine. Although the technology has quickly matured to enable nucleic acid sensing with transformative implications for genomics, biological pores stand as some of the most promising candidates to drive the next developments in single-molecule proteomics.

Targeting specific membranes with an azide derivative of the pore-forming peptide ceratotoxin A.

Pore-forming antimicrobial peptides (AMPs) are attracting interest as cytolytic antibiotics and drug delivery agents with potential use for targeting cancer cells or multidrug-resistant pathogens. Ceratotoxin A (CtxA) is an insect-derived cytolytic AMP with 36 amino acids that is thought to protect the eggs of the medfly Ceratitis capitata against pathogens. Single channel recordings using planar lipid bilayers have shown that CtxA forms pores with well-defined conductance states resembling those of alamethicin; it also forms one of the largest pores among the group of ceratotoxins. In this work, we modified CtxA at its N-terminus with an azide group and investigated its pore-forming characteristics in planar lipid bilayer experiments. We demonstrate the possibility to target specific lipids by carrying out click reactions in-situ on lipid membranes that display a dibenzocyclooctyne (DBCO) moiety on their head group. As a result of covalent linkage of the peptides to the bilayer, pore-formation occurs at 10-fold reduced peptide concentration and with a reduced dependence on the transmembrane voltage compared to unlinked CtxA-azide peptides or native CtxA peptides.

Single channel planar lipid bilayer recordings of the melittin variant MelP5.

MelP5 is a 26 amino acid peptide derived from melittin, the main active constituent of bee venom, with five amino acid replacements. The pore-forming activity of MelP5 in lipid membranes is attracting attention because MelP5 forms larger pores and induces dye leakage through liposome membranes at a lower concentration than melittin. Studies of MelP5 have so far focused on ensemble measurements of membrane leakage and impedance; here we extend this characterization with an electrophysiological comparison between MelP5 and melittin using planar lipid bilayer recordings. These experiments reveal that MelP5 pores in lipid membranes composed of 3:1 phosphatidylcholine:cholesterol consist of an average of 10 to 12 monomers compared to an average of 3 to 9 monomers for melittin. Both peptides form transient pores with dynamically varying conductance values similar to previous findings for melittin, but MelP5 occasionally also forms stable, well-defined pores with single channel conductance values that vary greatly and range from 50 to 3000pS in an electrolyte solution containing 100mM KCl.