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Single-molecule site-specific detection of protein phosphorylation with a nanopore


We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.

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Figure 1: Single-molecule nanopore detection of phosphorylation of a model substrate.
Figure 2: Single-molecule nanopore detection of four phosphorylation states.


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We thank E. Mikhailova for the preparation of α-hemolysin pores. We thank L. Harrington for the PKA-CAT plasmid and the purification protocol for the catalytic subunit of PKA. This work has been supported by the National Institutes of Health, Oxford Nanopore Technologies and an European Research Council Advanced Grant. C.B.R. is funded by the Danish National Research Foundation (grant number DNRF78) and Aarhus University, Faculty of Science and Technology.

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C.B.R. performed experiments, analyzed data and wrote the paper. D.R.-L. planned the research, performed experiments, analyzed data and wrote the paper. H.B. planned the research and wrote the paper.

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Correspondence to David Rodriguez-Larrea or Hagan Bayley.

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Competing interests

H.B. is the founder, and a director and shareholder of Oxford Nanopore Technologies, a company engaged in the development of nanopore sequencing technology. Work in the Bayley laboratory at the University of Oxford is supported in part by Oxford Nanopore Technologies.

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Rosen, C., Rodriguez-Larrea, D. & Bayley, H. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat Biotechnol 32, 179–181 (2014).

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