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Decoding Proteoforms with Single Acid Resolution Using a Sub-nanometer Diameter Pore

Lisauskas, T.; Rayabharam, A.; Murkate, P.; Almonte, L.; Rigo, E.; Robles, J. O.; Dong, Z.; Joseph, J.; Aluru, N. R.; Timp, G.

2022-12-22 biophysics
10.1101/2022.12.22.521660 bioRxiv
Show abstract

When a denatured protein isoform (i.e., a proteoform) immersed in electrolyte is impelled by an electric field through a sub-nanometer-diameter pore (i.e., a sub-nanopore) spanning a thin membrane, the sequence of amino acid (AA) residues constituting the proteoform can be directly "read" one at a time by measuring fluctuations in the electrolytic current. Corroborating this assertion, an analysis of the pore current with molecular dynamic (MD) simulations reveals that the fluctuations are correlated to the sequence of AA volumes, the water in the pore and affected by the acid mobility. After alignment to account for variations in the acid mobility, the simulated pore current is nearly perfectly correlated to the pattern of empirical fluctuations. To prove out the prospects for decoding proteoforms this way, site-specific post-translational modifications (PTMs) and point mutations in amyloid-beta (A{beta} 1-42) are analyzed with a sub-nanopore assay. The results show that single acids can be resolved in proteoforms with a dynamic range limited by the size of phenylalanine and glycine. With this sensitivity and single acid resolution, the sequence of a scrambled variant of A{beta} 1-42 was discriminated with a p-value < 10-5.

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