Proteins perform most cellular functions and are key biomarkers of health and disease, yet proteomic analysis lags far behind genomics due to the complexity and diversity of proteoforms. Recent advances in nanopore technology are transforming protein analysis. One promising strategy for protein identification is peptide profiling; however, its applicability remains limited to the identification of specific proteins, and discriminating among the vast variety of proteins is still challenging. Here, we propose a nanopore-based single amino acids (AAs) profiling method for protein identification. Combined with controlled enzymatic digestion, selected regions of the target proteins are hydrolyzed into individual AAs. We then conjugate a molecular tag to each released AAs to enhance its interactions with the nanopore interface, thereby amplifying the signal readouts, and improving the capture frequency. By reading corresponding electrical current signals of those AAs in a nanopore system, we thereby enable the robust construction of protein profiles. Our in silico prediction showed that with this method, it is sufficient to identify 91% of human proteins in the UniPort database. With future optimization, our results point to a new route for nanopore-based protein identification beyond peptide profiling.

Keywords: Protein identification; Amino acid profiling; Enzymatic digestion; Nanopore sensing