title: LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.
abstract: The GalNAc O-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 N- and O-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 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. 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. 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). 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.
authors.affiliation: Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Adnan
authors.initials: A
authors.lastname: Halim
authors.forename: Ulla
authors.initials: U
authors.lastname: Rüetschi
authors.forename: Göran
authors.initials: G
authors.lastname: Larson
authors.forename: Jonas
authors.initials: J
authors.lastname: Nilsson
mesh_terms: D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000737:chemistry; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D002853:Chromatography, Liquid; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006466:Hemopexin / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled; D012694:Serine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
chemical_list: D001057:Apolipoproteins E; C111572:GPR37L1 protein, human; D006023:Glycoproteins; D008565:Membrane Proteins; D011134:Polysaccharides; D043562:Receptors, G-Protein-Coupled; C478651:YIPF3 protein, human; D013912:Threonine; D012694:Serine; D006466:Hemopexin; D020794:Receptor Protein-Tyrosine Kinases; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase