Peptide sequencing via reverse translation of peptides into DNA

Abstract Scalable methods that can accurately sequence peptides at single-amino acid resolution could significantly advance proteomic studies. We present a protein sequencing method based on the “reverse translation” of peptide sequence information into DNA barcodes that document the identity, position, and the originating peptide of each amino acid. We employ a modified Edman degradation process that converts peptides into DNA-barcoded amino acids, which are subsequently detected by proximity extension assay, yielding multi-barcoded DNA outputs that can be PCR amplified and sequenced. Using our method, we sequenced multiple consecutive amino acids within a model peptide. This method also enables the differentiation of single amino acid substitutions, and the identification of post-translational modifications and their positions within multiple peptides simultaneously. With further development, we anticipate that this method will enable highly parallel de novo protein sequencing with single-molecule sensitivity.