Fluorosequencing without terminal residue cleavage

A modification to fluorosequencing, an emerging method for large-scale parallel protein identification, is proposed. In this modified approach terminal residues are not cleaved by Edman degradation (a key step in fluorosequencing) or any of its variants or enzymatic alternatives, the process is non-destructive. There are three parts: 1) a charged homopeptide header is attached to each of the peptides being partially sequenced; 2) the glass slide that the headers are immobilized on is mounted on a moveable platform in the cis chamber of an electrolytic cell (e-cell); and 3) header-peptides are advanced through an array of nanopores in the e-cell toward the trans chamber one residue at a time with a mechanical positioning system based on piezo-electric sensors and sub-nm movement precision. As in fluorosequencing, the optical signal from fluorescently tagged residues in the peptides is read with TIRF (Total Internal Reflection Fluorescence) microscopy. The difference here is that instead of repeatedly cleaving terminal amino acids (AAs), AAs are either revealed to the camera in sequence from the terminal AA (forward version) or hidden from it from the header end of the peptide (backward version). Computational simulations with nanopore diameter ~0.5-1 nm, pore lengths 3.5-7 nm, peptide lengths 10-20, and header lengths 20-45 suggest that with a long enough charged header the peptide can overcome diffusive dispersion so the terminal AA remains confined to a small neighborhood, making reliable tag readout possible. Additionally in the forward version if in each cycle tags are deactivated after they have been read and/or dissociated from their residues and washed away, signals take on a binary character and signal processing becomes more robust. This is an archival method in which the header-peptide can be moved in both directions and measurements can be done an arbitrary number of times while the sample remains intact.