PMID: 23234360

 

    Legend: Sugar

Title : LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins

Abstract :

1. The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective
2. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nano-LC- ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization
3. Here, we have introduced peptide N-glycosidase F ( PNGase F) pretreatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS(2 )/MS(3) search protocol for glycopeptide identification
4. 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-O- structure attached to one to four Ser/Thr residues
5. We characterized 106 O-glycosylations and found Pro residues preferentially in the n - 1, n + 1, and/or n + 3 positions in relation to the Ser/Thr attachment site (n)
6. 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