Top‐down tandem mass spectrometry on RN ase A and B using a Q h/ FT ‐ ICR hybrid mass spectrometer

Protein characterization using top‐down approaches emerged with advances in high‐resolution mass spectrometers and increased diversity of available activation modes: collision‐induced dissociation (CID), infrared multiphoton dissociation ( IRMPD ) electron capture dissociation ( ECD ), and electron transfer dissociation ( ETD ). Nevertheless, top‐down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top‐down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein R ibonuclease B ( RN ase B ). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RN ase B and its nonglycosylated analog RN ase A. The experiments were performed on a B ruker 12‐T Qh/FT‐ICR Solari X mass spectrometer using vibrational ( CID / IRMPD ) and radical activation ( ECD / ETD ) with/without pre‐ or post‐activation ( IRMPD or CID , respectively). The several activation modes yielded complementary sequence information. The radical activation modes yielded the most extensive sequence coverage that was slightly improved after a CID predissociation activation event. The combination of the data made it possible to obtain 90% final sequence coverage for RN ase A and 86% for RN ase B . Vibrational and radical activation modes showed high retention of the complete glycan moiety (>98% for ETD and ECD ) facilitating unambiguous assignment of the high‐mannose glycosylation site. Moreover, the presence of the high‐mannose glycan enhanced fragmentation around the glycosylation site.