PT  - JOURNAL ARTICLE
AU  - Tobias Ensslen
AU  - Kumar Sarthak
AU  - Aleksei Aksimentiev
AU  - Jan C. Behrends
TI  - Resolving isomeric posttranslational modifications using a nanopore
AID  - 10.1101/2021.11.28.470241
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.28.470241
4099  - http://biorxiv.org/content/early/2021/11/28/2021.11.28.470241.short
4100  - http://biorxiv.org/content/early/2021/11/28/2021.11.28.470241.full
AB  - 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 sequencing1. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems2–4. 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 target5,6. 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.Competing Interest StatementJ.C.B. is co-founder and shareholder of Nanion Technologies GmbH, Munich, Germany and Ionera Technologies GmbH, Freiburg, Germany