Disambiguation of Product Ion Assignments, Including Internal Fragment Ions from Top-Down Mass Spectrometry, Using...

Advances in mass spectrometry instrumentation resulted in richer MSn spectra and additional ambiguity in peak assignments. Internal fragment ions (IFs) are often prevalent during top-down mass spectrometry (TDMS) and are particularly difficult to assign─to the extent many practitioners currently forgo their assignment. Accounting for IFs during high-yield dissociation can increase the depth of sequence coverage by an order of magnitude but also results in a geometric increase in the size of the database and ambiguous peak assignments. The ambiguity in TDMS fragment ion assignments comes from the potential for one experimental peak to be assigned to two or more theoretical molecules─most often in the form of isomeric internal fragment ions from distinct sequence regions. The disambiguation of such peaks remains a major challenge in TDMS and is the focus of this work. Ion mobility (IM) mass spectrometry can often separate isomers, including peptides. Starting from a TDMS spectrum containing both unambiguously and ambiguously assigned peaks, we address the hypothesis that ion mobility, or ion mobility followed by fragmentation, can help disambiguate fragment ion assignments by resolving isomeric ambiguity in the mobility domain. We demonstrate that mobility-aligned fragmentation (MAF) can disambiguate TDMS and peptide MS/MS peak assignments of isomeric terminal and internal ions. Consistent with previous studies, we also demonstrate that a single isomeric species (e.g., an individual b-, a-, or internal ion) can be comprised of multiple conformational isomers with different collisional cross-section (CCS) values.