Fluorosequencing without Edman degradation

Abstract

A modification to fluorosequencing, an emerging method for large-scale parallel protein identification, is proposed. In this modified approach Edman degradation (ED), a key step in fluorosequencing, is avoided. There are three parts: 1) attaching a charged homopeptide header to each of the peptides being partially sequenced; 2) mounting the glass slide that the peptides are bound to on a moveable platform in the cis chamber of an electrolytic cell (e-cell); and 3) advancing the header-peptides 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 applying ED to 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 and the terminal AA remains confined to a small neighborhood to make reliable tag readout possible. Additionally in the forward version if in each cycle tags are dissociated from their residues after they have been read and then washed away, signals take on a binary character, which makes signal processing more robust. This is a non-destructive 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.