PMID: 25456591

 

    Legend: Gene, Sites

Title : Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift

Abstract :
  1. We reported an improved combinatorial approach for identifying site-specific O-glycosylation using both glycan cleaved and non-cleaved methods
  2. In this approach, a non-reducing β-elimination kit coupled with non-specific enzymes performed efficient digestion, O-glycan cleavage, and partial dephosphorylation without significant side reactions, thus enabling an automatic database search for the cleaved O-glycosylation or serine/threonine (S/T) phosphorylation sites
  3. From the same sample concurrently prepared without β-elimination, the corresponding intact O-glycopeptides were mapped by accurate precursor ion mass using an in-house glycan database majorly composed of GalNAc (mucin-type) core and the retention-time shift (ΔRt)
  4. Each glycopeptide assignment was verified by the detection of glycan-specific fragments using collision-induced dissociation (CID) to estimate False Discovery Rate (FDR)
  5. Using fetuin as a model, all identified S/T elimination sites were matched to multiple intact glycopeptides with a 31% FDR
  6. This considerably reduced to 0% FDR by ΔRt filtering
  7. This approach was then applied to a protein mixture composed of therapeutic Factor IX and Enbrel(®) mixed with fetuin and kappa-casein
  8. A total of 26 glycosylation sites each of which corresponds to 1-4 glycans were positively mapped and confirmed
  9. The FDR decreased from 33% to 3.3% by ΔRt filtering and exclusion of repeated peptide tags that covered the same glycosylation sites
  10. Moreover, the phosphorylation and O-glycosylation on the same site such as T159 of Factor IX and T170 of kappa-casein were able to be unambiguously differentiated
  11. Thus, our approach is useful for in-depth characterization of site-specific O-glycosylation of a simple mixture such as protein-based therapeutics
Output (sent_index, trigger, protein, sugar, site):
  • 0. O-glycosylation, , -, -, site
  • 10. O-glycosylation, , -, -, site
  • 3. O-glycopeptides, , -, -, O-glycopeptides
  • 4. glycopeptide, , -, -, glycopeptide
  • 5. glycopeptides, , -, -, glycopeptides
  • 8. glycosylation, , -, -, sites
  • 9. glycosylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX