Terminal tagging of full-length proteins for enhanced capturing by biological nanopores

Having demonstrated protein fingerprinting and a strong potential for single-molecule protein sequencing, nanopores have found success in a broad range of protein translocation experiments in the past years. Most analytes tested thus far have been engineered proteins or peptide analytes, due to such limitations of native proteins as non-uniform electric charge along the chain and heterogeneity of conformation of the terminal motifs, both affecting the efficiency of capture and threading of the analyte. While this can be mitigated by engineering of the nanopore, a universal solution that normalizes sample preparation for protein analytes is still being sought by researchers in the field. Here, we show enhanced translocation of native proteins through biological nanopores by chemically tagging the N-termini of proteins with a densely charged motif such as DNA and peptide oligomers. The major challenge of such tagging chemistry is the off-target modification on the primary amines of lysine sidechains. We tested various tagging chemistry, and as expected, nanopores are susceptible to clogging from those products with lysine sidechain off-target tagging. Thus, we tuned our method to reduce such effect. We compared the capture rate and translocation signal of pristine and tagged native proteins, showing an enhancement in capture and translocation. We also compared the translocation of the tagged and the engineered versions of a protein for evidence of successful translocation. We tested several native proteins of distinct origin to show the universality of our method. Our method is generalizable to diverse architectures for protein translocation through nanopores, which will accelerate the implementation of the potential of nanopore in proteomics and unlock the reservoir of native proteins from organisms to researchers in this field.