Internal Fragment Ions Disambiguate and Increase Identifications in Top-Down Proteomics.

A large fraction of observed fragment ion intensity remains unidentified in top-down proteomics. The elucidation of these unknown fragment ions could enable researchers to identify additional proteoforms and reduce proteoform ambiguity in their analyses. Internal fragment ions have received considerable attention as a major source of these unidentified fragment ions. Internal fragments are product ions that contain neither protein terminus, in contrast with terminal ions that contain a single terminus. There are many more possible internal fragments than terminal fragments, and the resulting computational complexity has historically limited the application of internal fragment ions to low-complexity samples containing only one or a few proteins of interest. We implemented internal fragment ion functionality in MetaMorpheus to allow the proteome-wide annotation of internal fragment ions. MetaMorpheus first uses terminal fragment ions to identify putative proteoforms and then employs internal fragment ions to disambiguate similar proteoforms. In the analysis of mammalian cell lysates, we found that MetaMorpheus could disambiguate over half of its previously ambiguous proteoforms while also providing up to a 7% increase in proteoform-spectrum matches identified at a 1% false discovery rate.