A Universal-Binder, De Novo Peptide Sequencing Platform with a Fluorescence Lifetime Fingerprint

The recent global push towards next-generation protein sequencing has resulted in powerful mass spectrometry and fluorescence-based approaches; however these technologies are limited in their ability to completely sequence proteins de novo, suffer from low throughput, and lack the sensitivity to interpret post-translational modifications with high fidelity. To address these issues, a single-molecule peptide sequencing method was developed to utilize a universal binder approach with an optical readout. In our established workflow, peptides were bound to a glass surface by the C-terminus, leaving a primary amine at the N-terminus for covalent attachment of a phenyl-isothiocyanate-functionalized oligonucleotide. A fluorophore-conjugated complementary imaging strand was hybridized with the docked oligo to bring a fluorophore near to the N-terminal amino acid and allow imaging by two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to collect fluorescence lifetime fingerprints. We demonstrate that there are preferential interactions among 4 separate fluorophores and 12 amino acids, including 2 that are post-translationally modified, that lead to detectable changes in the fluorescence lifetime. We further highlight the sequencing capability by performing multiple Edman Degradation cycles while collecting fluorescence lifetime fingerprints from each amino acid during the process. Development of this technology can provide a high sensitivity, high throughput method to sequence peptides, without the use of amino acid-specific binders.