Detecting protein post-translational modifications with nanopores

Proteins play a crucial role in various cellular functions, from providing structural support to catalyzing chemical reactions. Following their synthesis, many proteins undergo a series of biochemical transformations that increase their complexity and functionality. These transformations, known as Post-Translational Modifications (PTMs), are critical for regulating protein activity, structure, localization, and interactions. For instance, phosphorylation can either activate or deactivate enzymes; similarly, glycosylation impacts protein stability and localization.

Defects in PTMs have been linked to numerous human diseases, and therefore, characterizing the changes in PTM types and levels is highly relevant for early diagnosis and monitoring the progression of diseases.

Despite their significance, detecting and analyzing PTMs is challenging. Traditional methods like mass spectrometry and antibody-based assays face limitations, particularly when detecting multiple modifications on a single protein molecule or analyzing low-abundance protein PTMs.

Recent advances have opened up a new frontier in the study of PTMs: nanopore technology. Traditionally used in DNA sequencing, this technology is now being adapted to study proteins and their modifications. Compared to other analytical methods, nanopore-based approach is faster, more cost-effective, and offers single-molecule resolution.

In a recent study, scientists from Ecole Polytechnique Fédérale de Lausanne showcased the use of a biological nanopore, the pore-forming toxin aerolysin, for detecting α-synuclein-derived peptides with single or multiple PTMs. This method proved sensitive enough to identify PTMs such as phosphorylation, nitration, and oxidation in various combinations, even at low protein concentrations (in the picomolar range).

α-synuclein was selected as a model system because of the strong link between its PTM profile and several neurodegenerative diseases, such as Parkinson’s disease. The authors performed nanopore single-channel recordings (using Orbit mini device) and examined how different α-synuclein-derived peptides affected ionic currents through nanopores under an applied potential. Notably, modifications like phosphorylation, nitration, and oxidation significantly altered the current amplitude and dwell time compared to wild-type α-synuclein. Additionally, peptides with multiple PTMs and different combinations produced distinct ionic current signatures, which could be confidently identified using a deep learning model for signal processing.

Overall, this study highlights the potential of nanopore sensing in PTM detection, emphasizing its applicability in biomarker discovery, diagnostics, and single-molecule proteomics. Nanopore-based protein analysis offers a faster, more affordable, and label-free alternative to traditional methods, paving the way for the development of portable diagnostic devices.

For more details, please refer to the paper here: https://pubs.acs.org/doi/10.1021/acsnano.3c08623

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