Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology.
Bibliographical noteFunding Information:
We would like to thank Peggy Bohländer, Rienk Eelkema, Martin Pabs, Robert Cordfunke, Sonja Schmid, and Mike Filius for fruitful discussions. This work was supported by the Foundation for Fundamental Research on Matter (Vrije Programma SMPS) to C.J., C.D., and G.M., and by the European Research Council Advanced Grant SynDiv (No. 669598), The Netherlands Organisation for Scientific Research (NWO/OCW) through the NanoFront and BaSyC grants
Copyright © 2019 American Chemical Society.
- label-free detection
- post-translational modifications