Protein glycosylation is a critical post-translational modification (PTM) that affects protein folding and stability. It is also essential for cell-cell adhesion and, as such, plays a role in immune response, cancer, and numerous diseases. Mass spectrometry (MS) instrument sensitivity is one of the significant limitations when analyzing glycopeptides due to the heterogeneity of glycan structures, resulting in multiple peptide isoforms with much lower abundances than their non-glycosylated forms. The challenges associated with low glycopeptide abundances can be overcome with strategies like enrichment of glycopeptides, a lower flow rate LC separation regime (i.e., nanoflow LC separation), and high-performance MS systems for glycopeptide detection and characterization.

Another challenge for glycopeptide characterization comes from the labile nature of the glycosylation modification on peptides. Common fragmentation methods such as collision-induced dissociation (CID) can provide peptide backbone information but often result in fragments lacking the labile side chain modifications. Alternative fragmentation methods, such as electron activated dissociation (EAD)-based MS/MS, have been shown to provide site-specific PTM localization due to the retention of these modifications on the resulting fragment ions.

In this work, nanoflow LC separation and EAD fragmentation were used to demonstrate the identification of N-glycopeptides in depleted, digested and glycopeptide-enriched human plasma. Using 60-minute nanoflow LC gradients, EAD-based data-dependent acquisition (DDA), and data processing with PEAKS GlycanFinder software, >1000 glycopeptides were identified from samples derived from 37.5 µg of plasma.