Spectral Quality-Guided Optimization of Hybrid EAciD Fragmentation on the ZenoTOF 7600 System Enables Efficient Characterization of the Serum N‑Glycoproteome

The analysis of N-glycopeptides using mass spectrometry comes with numerous challenges, one of which is the unique fragmentation features associated with glycopeptides. Generally, low energy collision-based fragmentation can provide valuable information by the glycan-related B- and Y-ions but often at the expense of peptide backbone coverage. On the other hand, while electron-based fragmentation methods provide good peptide backbone coverage and allow glycan localization, they do not generate sufficient structural details on the glycans by missing B- and Y-ions. Here, we systematically optimized a ZenoTOF 7600 system employing electron-activated dissociation (EAD) as an alternative electron-based fragmentation method. Combined with collision-induced dissociation (CID), hybrid fragmentation (EAciD) on N-glycopeptides generated information-rich MS2 spectra that improved the identification of N-glycopeptides in complex samples. We applied this method to a small cohort of blood donors, analyzing glycopeptide-enriched blood samples of healthy donors and patients suffering from pancreatic cancer or hepatocellular carcinoma. On the ZenoTOF 7600 system, we were able to efficiently detect ∼1900 glycopeptides per sample. EAciD especially performed well in sequencing and identifying very large N-glycopeptides carrying sizable and structurally complex glycan moieties. This allowed us to observe disease-specific N-glycopeptide signatures on numerous highly abundant blood proteins, mostly related to changes in fucosylation and complex extended glycoforms.