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

J Proteome Res. 2013 Feb 1;12(2):573-84. doi: 10.1021/pr300963h. Epub 2013 Jan 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apolipoproteins E / chemistry
  • Cerebrospinal Fluid / chemistry*
  • Chromatography, Liquid
  • Glycomics
  • Glycoproteins / chemistry*
  • Glycosylation
  • Hemopexin / chemistry
  • Humans
  • Membrane Proteins / chemistry
  • Molecular Conformation
  • Molecular Sequence Data
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / chemistry
  • Polysaccharides / chemistry*
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptors, G-Protein-Coupled
  • Serine / chemistry*
  • Spectrometry, Mass, Electrospray Ionization
  • Threonine / chemistry*
  • Trypsin / chemistry


  • Apolipoproteins E
  • GPR37L1 protein, human
  • Glycoproteins
  • Membrane Proteins
  • Polysaccharides
  • Receptors, G-Protein-Coupled
  • YIPF3 protein, human
  • Threonine
  • Serine
  • Hemopexin
  • Receptor Protein-Tyrosine Kinases
  • Trypsin
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase