PMID: PMC6243375-1-1

 

    Legend: Gene, Sites

Title : Extraction of site‐specific O‐linked glycopeptides

Abstract :
  1. In EXoO, proteins are first digested to generate peptides, which are then conjugated to a solid support
  2. After washing, the O‐linked glycopeptides are enzymatically released from the support using an endo‐protease OpeRATOR that requires the presence of O‐linked glycans to specifically cleave on the N‐terminal side of O‐linked glycan‐occupied Ser or Thr (Fig 1A)
  3. To demonstrate proof of principle, bovine fetuin was analyzed and the six known O‐linked glycosylation sites documented in the UniProt database were pinpointed at Ser‐271, Thr‐280 , Ser‐282, Ser‐296, Thr‐334, and Ser‐341 (Dataset EV1)
  4. In addition, a new O‐linked glycosylation site at Ser‐290 was also identified (Dataset EV1 and Appendix Fig S1)
  5. Of note, O‐linked glycans were still attached to the site‐specific O‐linked glycopeptides as confirmed by the detection of oxonium, peptide (Y0), and less commonly identified peptide + HexNAc (Y1) ions in the MS/MS spectrum (Fig 1B)
  6. The detection of oxonium ions in the MS/MS spectrum is particularly useful for obtaining the correct identification of O‐linked glycopeptides
  7. In addition, the chemical conjugation of peptides to a solid support allows efficient washing and specific enzymatic release of intact O‐linked glycopeptides
  8. As a result, 193 peptide spectrum matches (PSMs) were assigned to fetuin site‐specific O‐linked glycopeptides with Ser or Thr at the N‐termini of peptides , glycan modification, and oxonium ions in the MS/MS spectra from a total of 270 assigned PSMs, indicating a specificity of approximately 71.5% for O‐linked glycopeptide enrichment using EXoO (Dataset EV1)
  9. The analysis of fetuin demonstrated the ability of EXoO to enrich and identify O‐linked glycopeptides at specific O‐linked glycosylation sites and their corresponding O‐linked glycans
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycopeptides, , -, -, glycopeptides
  • 2. glycopeptides, , -, -, glycopeptides
  • 3. glycosylation, , -, -, sites
  • 4. glycosylation, , -, -, Ser‐290
  • 4. glycosylation, , -, -, site
  • 5. glycopeptides, , -, -, glycopeptides
  • 6. glycopeptides, , -, -, glycopeptides
  • 7. glycopeptides, , -, -, glycopeptides
  • 8. glycopeptide, , -, -, glycopeptide
  • 8. glycopeptides, , -, -, glycopeptides
  • 9. glycopeptides, , -, -, glycopeptides
  • 9. glycosylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
  • 8. fetuin, glycopeptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX