Historically, this question was initially answered via laborious, manual annotation of individual MS2 spectra. In this approach, a human expert would examine an MS2 spectrum, searching for pairs of peaks whose mass difference corresponds to that of an amino acid. If the peptide fragmented fairly uniformly along its backbone, it might be possible to identify a series of such peak pairs and thereby reconstruct the full peptide sequence. Furthermore, experts leveraged their own set of additional, internalized rules-such as the suppression of a b-ion's intensity when followed by proline-to resolve ambiguities and enhance confidence in their sequence assignments . Early algorithms for de novo sequencing codified this process into algorithmic procedures .