Trypsin first cuts a protein into short peptides. Inside the mass spectrometer, each peptide is fragmented a second time at every backbone bond, producing complementary b-ion (N-terminal) and y-ion (C-terminal) fragments whose masses depend on the exact residue order — this is the peptide's "fingerprint" spectrum.
- Fragment sample — breaks the selected peptide at each bond in turn and plots every resulting ion mass as a peak: the observed spectrum.
- Run database search — predicts the same kind of spectrum for each candidate reference peptide and checks, peak by peak, how many predicted masses land within tolerance of an observed peak.
- Match tolerance — the mass window (Da) used to call two peaks "the same"; real instruments use a much tighter ppm window set by their analyser resolution.
Two peptides can share the same total mass (an anagram of residues) yet fragment completely differently — which is why sequence identification needs the whole peak pattern, not just the parent mass.