posted on 2024-01-11, 17:03authored byRuiqing Li, Chaoshuang Xia, Shuye Wu, Margaret J. Downs, Haowei Tong, Nafisa Tursumamat, Joseph Zaia, Catherine E. Costello, Cheng Lin, Juan Wei
Glycosylation is widely recognized as the most complex
post-translational
modification due to the widespread presence of macro- and microheterogeneities,
wherein its biological consequence is closely related to both the
glycosylation sites and the glycan fine structures. Yet, efficient
site-specific detailed glycan characterization remains a significant
analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform,
higher-energy electron-activated dissociation (heExD) tandem mass
spectrometry (MS/MS) revealed extraordinary efficacy for the structural
characterization of intact glycopeptides. HeExD produced extensive
fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions
from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments
(with or without the glycan). The intensity of cross-ring cleavage
and backbone fragments retaining the intact glycan was highly dependent
on the electron energy. Among the four electron energy levels investigated,
electronic excitation dissociation (EED) provided the most comprehensive
structural information, yielding a complete series of glycosidic fragments
for accurate glycan topology determination, a wealth of cross-ring
fragments for linkage definition, and the most extensive peptide backbone
fragments for accurate peptide sequencing and glycosylation site localization.
The glycan fragments observed in the EED spectrum correlated well
with the fragmentation patterns observed in EED MS/MS of the released
glycans. The advantages of EED over higher-energy collisional dissociation
(HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy
collisional dissociation (EThcD) were demonstrated for the characterization
of a glycopeptide bearing a biantennary disialylated glycan. EED can
produce a complete peptide backbone and glycan sequence coverage even
for doubly protonated precursors. The exceptional performance of heExD
MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization
on an Orbitrap-Omnitrap hybrid instrument presents a novel option
for in-depth glycosylation analysis.