LC–MS/MS Characterization of O‑Glycosylation Sites and Glycan Structures of Human Cerebrospinal Fluid Glycoproteins

The GalNAc <i>O</i>-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic <i>N</i>- and <i>O</i>-glycopeptides from human cerebrospinal fluid glycoproteins using nano-LC–ESI–MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide <i>N</i>-glycosidase F (PNGase F) pretreatment of CSF samples to remove the <i>N</i>-glycans facilitating the selective characterization of <i>O</i>-glycopeptides and enabling the use of an automated CID–MS²/MS³ search protocol for glycopeptide identification. We used electron-capture and -transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core-1-like HexHexNAc-<i>O</i>- structure attached to one to four Ser/Thr residues. We characterized 106 <i>O</i>-glycosylations and found Pro residues preferentially in the <i>n</i> – 1, <i>n</i> + 1, and/or <i>n</i> + 3 positions in relation to the Ser/Thr attachment site (<i>n</i>). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease