Peptide biomarker enantiomer discrimination and identification of a biomarker family by nanopore in a complex biofluid

There is a current need to develop methods for sensitively detecting peptide biomarkers in complex biofluids to enable early disease detection. Moreover, to our knowledge, no detection method is currently capable of identifying the different conformations of peptide biomarkers differing by a single amino acid, or L- and D-peptide enantiomers. Single-molecule nanopore sensing promises to provide this level of resolution. To identify these differences in a complex biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed. We are interested here in two families of peptide biomarkers differing by a single amino acid (depleted or chiral amino-acid substitution) involved in normal biological processes and many disabling pathologies. We show the proof of concept for (1) directly identifying a family of biomarkers in serum and (2) discriminating biomarker enantiomers at the single molecule level using a protein nanopore. Each peptide exhibits unique electrical signatures attributed to specific conformations in bulk. To extend the utility of our experimental results, we developed a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used semi-supervised classification to assign each event type to a specific biomarker.