Photo-Fragmentation Methods for Single-Molecule Protein Sequencing by Nanopore Mass Spectrometry

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PROJECT SUMMARY A single molecule protein sequencing technology would revolutionize our ability to study biology and to diagnose and treat disease. Mass spectrometry has a unique ability to identify amino acids by their mass-to-charge ratios, but current instruments fall well short of the single-molecule sensitivity limit. On the other hand, nanopore sensors operate on single molecules and naturally preserve the sequential ordering of a biopolymer, but they cannot discriminate the different amino acids. We envision a protein sequencing technology in which a new, nanopore-based ion source addresses the current shortcomings of mass spectrometry. The basic idea to first denature the protein, then drive it through a nanoscale hole that compels its constituent amino acids to pass in sequential order, then break the polymer into separate monomers by photo-fragmentation, and finally deliver those amino acids into a mass spectrometer where they can be identified by their mass-to-charge ratios. We recently demonstrated a nanopore ion source that can deliver desolvated amino acid ions directly into the high vacuum part of a mass spectrometer from aqueous solution. The nanopore ion source thus circumvents the sample loss mechanisms inherent to conventional electrospray ionization (ESI), where charged droplets are sprayed into a background gas that scatters ions and degrades their transmission. Furthermore, we recently designed a mass spectrometer that exhibits two new capabilities we need for sequencing: 1) an ability to simultaneously measure ions with different masses and 2) an ability to tag each ion with an arrival time with sub-100 ns temporal resolution. The main unproven part of our sequencing strategy is a method for transforming peptides (i.e., polymers) into separate amino acids inside the nanopore ion source. This project will investigate the feasibility of fragmenting peptides with light within a nanopore ion source. We seek to fragment peptides into separate but intact amino acids. Ultraviolet light with a wavelength near 200 nm is promising for this purpose because it is selectively absorbed by peptide bonds and because a single photon carries enough energy to induce scission of that bond. Through the Aims of this project, we will measure the speed and selectivity with which different wavelengths of light cleave the peptide bond, and we will apply that knowledge to demonstrate peptide fragmentation within our nanopore ion source.