Complete Top-down Sequencing of Therapeutic Phosphorodiamidate Morpholino Oligomers by Multimodal Ion Activation in Both Polarities

Phosphorodiamidate morpholino oligomers (PMOs) are an important class of approved antisense therapeutics whose neutral backbone presents significant challenges for tandem mass spectrometric sequencing. Here, we evaluate multiple ion activation strategies on a timsOmni™ prototype in both electrospray polarities using the approved PMO drugs viltolarsen (21-mer) and eteplirsen (30-mer). In negative ion mode, resonance collisioninduced dissociation (RCID) and infrared multiphoton dissociation (IRMPD) each provided complete sequence coverage for both PMOs from charge states generated under standard infusion conditions. Electron detachment dissociation followed by RCID (EDD-RCID, MS3) also achieved full coverage of viltolarsen and produced diagnostic d- and z-type ions that retained the labile dimethylamino group, which was lost during purely vibrational activation. In positive ion mode, conventional CID resulted primarily in nucleobase loss with no backbone cleavage. However, electron-based activation methods, including electron capture dissociation (ECD) and electron-induced dissociation (EID), combined with supplementary RCID of the radical cation, restored backbone fragmentation and enabled complete sequence coverage. Propylene carbonate (PC) proved valuable to achieve complete coverage by generating the higher charge states required for EDD-RCID and positive-mode electron-based workflows. Together, these results outline several effective activation strategies for PMO sequencing.