Selective fragmentation of the N‐glycan moiety and protein backbone of ribonuclease B on an Orbitrap Fusion Lumos Tribrid mass spectrometer.

Rationale: The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored. Methods: RNase B solution was electrosprayed and its intact glycoforms of GlcNAc2Mann (n = 5–9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N‐glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N‐glycan database search engines ProteinGoggle and GlySeeker, respectively. Results: With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N‐glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods. Conclusions: For comprehensive structural characterization of intact N‐glycoproteins using tandem mass spectrometry, the composition and topology of the N‐glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters. [ABSTRACT FROM AUTHOR]