Reference-free protein sequencing by consensus assembly of redundant de novo peptide reads

Reading a protein’s sequence from tandem mass spectra without a reference is limited by single-spectrum accuracy, most acutely across the hypervariable complementaritydetermining regions of antibodies. Broadly specific proteases tile a protein with long, overlapping peptides, so every residue is covered by many independent de novo reads. borgonovo assembles their per-step probability profiles into a reference-free per-residue consensus, seeding templates from mass-closure-consistent reads and recruiting the rest by substitution- tolerant alignment and per-column voting. Re-decoding each spectrum with a prior from its consensus position lifts amino acid accuracy on placed spectra from 0.80 to 0.87. On the therapeutic antibody trastuzumab, nine proteases cover its heavy and light chains completely at 0.88 fixed-window identity, and 0.93 on the pruned assembly once local indels are accommodated. Applied unchanged to five secretome proteins and trastuzumab with three proteases, it reaches 0.87 mean fixed-window identity over 82% coverage. borgonovo is open source and works with most de novo sequencers, so redundant digestion turns any of them into a protein sequencer where no reference exists.