Resolving isomeric posttranslational modifications using a nanopore

Abstract

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.

Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore

“…Thus, the addition of a single methylene group to the side chain of an amino acid produces a small yet measurable decrease of the blockade current and the magnitude of such a decrease is quantitatively similar to the reduction of the nanopore conductive volume. Thus, steric exclusion determines the blockade current not only in the socalled whole molecule sensing measurements, 26,36 but also in strand sequencing of a polypeptide, a conjecture echoed by recent experiments. 37,38 The steric footprint is found to introduce a small yet statistically significant difference in the effective force acing on the polypeptides, with the bulkier side chains reducing the effective force, Figure 3f.…”

Precise Structural Analysis of Neutral Glycans Using Aerolysin Mutant T240R Nanopore

“…To gain deeper insights into the movement of molecules within the nanopore, an all-atom molecular dynamics (MD) approach was utilized to simulate the transport of tagged glycan molecules through the mutant T240R nanopore. 52,53 The mutant T240R was derived from the initial all-atom model of WT AeL (obtained from the Protein Data Bank, PDB ID: 5JZT) using the VMD Mutator plugin. 27 The construction of the molecules R-BN-Gb4 and R-BN-iGb4 was carried out using Gaussian software, 54 and their characteristics were described by the CHARMM36 force field 55 through the CGenFF Web server.…”

Volumetric analysis of unmodified and modified amino acids and its application to the detection of post-translational modifications (PTMs) with a nanopore

“…In [31] differences in blockade level and dwell time are used to identify a PTM in a protein without using labels. In [32] molecular dynamics simulations are used to show that acetylated and methylated residues in an intact peptide from histone protein H4 can be detected based on differences in the electrical resistance measured during passage of the peptide through the pore.…”