• Orbit 16 TC

    fully parallel recording of 16 bilayers

2021 - Resolving isomeric posttranslational modifications using a nanopore

Icon Orbit  Orbit 16 pre-print publication in bioRxiv (2021)

Ensslen T., Sarthak K., Aksimentiev A., Behrends J.C.,

bioRxiv (2021) doi: 10.1101/2021.11.28.470241


Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pore’s sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pore’s sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.

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