Statistical Considerations for the Assignment of Internal Ions in Top‐Down Mass Spectrometry

Top‐down mass spectrometry is a widely used tool for analyzing intact proteins to characterize proteoforms and identify post‐translational modifications. Frequently, fragmentation is required for full characterization, producing highly complex datasets. Despite best efforts, many peaks in top‐down datasets remain unassigned, which has prompted investigation of internal fragment ions. Internal ions can occur if the protein backbone is cleaved twice, producing a fragment that contains neither terminus. However, because the number of potential internal fragments greatly exceeds that of terminal ions, their assignment may be prone to false positives due to overlapping masses. In this article, we used higher‐energy collisional dissociation data from four proteins of increasing size to investigate the propensity for false‐positive assignments of internal fragments against shuffled sequences. We also evaluated the inherent overlap in internal fragment‐ion space between a given sequence and a shuffled protein of the same size. The propensity for matching assignments multiple times at the same residue was also determined for true and shuffled sequences. Overall, our findings reveal that under conditions typically employed for top‐down experiments, the extremely high number of potential internal ions creates unacceptable uncertainty for confident assignment.