Discrimination of cationic peptides using malaria translocon, EXP2, nanopores

Protein identification provides crucial information on the extensive biochemical reaction in our life. Although mass spectrometry has generally been used for protein/peptide identification, the scarcity of proteins in biological samples and post-translational modifications have remained a challenge. Nanopores, which provide rapid and sensitive single-molecule analysis have attracted attention as an alternative to these techniques. The sensing accuracy of nanopores mainly depends on the congeniality of electrostatic affinity and size between the nanopores and molecules. In this study, we evaluated the capability of peptide detection on EXP2 nanopore, which intrinsically transports a polypeptide chain in vivo as a malaria translocon, using peptides with different sizes and numbers of charges. Our previous studies found that wild-typed EXP2 (Wt-EXP2) nanopore could detect and identify two types of poly-L-lysine (short poly-L-lysine: 10,000 Da, long poly-L-lysine: 30,000-70,000 Da) with different molecular weights. Although the Wt-EXP2 nanopore could discriminate the poly-cationic peptides, the inherent current noise of the Wt-EXP2 nanopore in the analyte-free state interfered with more accurate single-molecule detection. This current noise probably owing to the fluctuation of its C-terminus region, so we designed ΔD231-EXP2, a variant with the deletion of the C-terminus region. The result shows that the current noise frequency of ΔD231-EXP2 nanopore decreased by half compared with Wt-EXP2, and we succeeded in discriminating the two types of oligo-arginine (R7X, X=W, G). Furthermore, the capture frequencies of these cationic peptides of the EXP2 nanopore were higher than the α-hemolysin nanopore. ΔD231-EXP2 nanopore also could identify the five types of trypsinized fragments (893.4-1,429 Da) from lysozyme. These results suggest that the EXP2 nanopore may be suitable for cationic peptides detection and identification. We are going to apply another variant EXP2NC (K42D+S46F) whose amino acid in the constriction region is exchanged for more accurate peptide identification.