PMID: PMC6243375-1-3

 

    Legend: Gene, Sites

Title : Specificities of OpeRATOR for peptides and O‐linked glycans

Abstract :
  1. To define cleavage specificities of peptides and O‐linked glycans of OpeRATOR in the complex samples, sequential ETD/HCD ‐ MS2 analysis was conducted on serum O‐linked glycopeptides generated using the EXoO method
  2. With 1% FDR, HCD ‐ MS2 and ETD‐MS2 identified 85 and 40 unique intact glycopeptides , respectively, with 27 glycopeptides identified in both modes (Dataset EV5)
  3. Next, the ambiguity of O‐linked glycosylation site at the first amino acid position on glycopeptides was determined in the PSMs generated by ETD‐MS2
  4. Among the 113 PSMs from ETD‐MS2 analysis, 105 PSMs were assigned to have core 1 O‐linked glycans conjugated at glycosylation sites localized at the first amino acid position of glycopeptides with ptmRS site probabilities of over 99% (Dataset EV5)
  5. O‐linked glycosylation sites with core 1 O‐linked glycans in seven PSMs could not be located by ETD spectra (Dataset EV5)
  6. One PSM was identified to have the O‐linked glycosylation site assigned at the sixth amino acid position on a serine (Dataset EV5)
  7. However, a second PSM of the same precursor was assigned to the first threonine residue (Dataset EV5)
  8. The observation of two PSMs of the same precursor with different site localization suggested that the site localization for this glycopeptide might not be confident for site assignment
  9. Therefore, ETD‐MS2 provided that a cleavage specificity by OpeRATOR was at the N‐termini of the O‐linked glycosylation sites with core 1 glycans
  10. HCD ‐ MS2 appeared to identify more unique glycopeptides compared to ETD‐MS2 that might be due to shorter glycopeptides with low charge states generated by trypsin and OpeRATOR digestion, while glycopeptides identified by ETD‐MS2 contained only +3 charge and above (Dataset EV5)
  11. Therefore, one of the advantages of EXoO method for O‐linked glycoproteomics analysis empowered efficient O‐linked glycosylation site localization by high cleavage specificity of OpeRATOR that was confirmed by ETD‐MS2
  12. The precise specificity of OpeRATOR for different O‐linked glycans remains unclear
  13. Analysis of our data from tissue, serum, and cells revealed that approximately 69% of total PSM contained glycan com position Hex(1)HexNAc(1) that was most likely to be core 1 mucin‐type glycan Gal‐GalNAc
  14. Therefore, it is possible to define that the O‐linked glycopeptide contained Hex(1)HexNAc(1) or most likely to be Gal‐GalNAc with or without sialic acid at the site of O‐linked glycosylation
  15. These data could also be explained as that the major glycan com position for site‐specific O‐linked glycopeptide is the core 1 structure Hex(1)HexNAc(1) that is prevalent in a wide range of glycoproteins from different cell types compared to the relatively restricted presence of other core structures seen in specific tissue and cell types (Brockhausen & Stanley, 2015)
  16. However, the fact that other glycoforms accounted for approximately 31% of total identified glycan com positions argues that further investigation is needed to definitively establish the glycoform specificity of OpeRATOR
  17. In addition, the possibility of multiple glycans on a glycopeptide demands caution in the data interpretation to define site‐specific glycan com position
  18. For example, two Hex(1)HexNAc(1) on a glycopeptide could yield a glycan com position of Hex(2)HexNAc(2) in the result
  19. EXoO may miss O‐linked glycosylation sites that are not in an appropriate peptide length for identification
  20. It can be anticipated that using enzymes other than trypsin for generating peptides will increase the identification of O‐linked glycosylation sites (Choudhary et al, 2003)
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycopeptides, , -, -, glycopeptides
  • 10. glycopeptides, , -, -, glycopeptides
  • 11. glycosylation, , -, -, site
  • 13. composition, , -, glycan composition Hex, position
  • 14. glycopeptide, , -, -, glycopeptide
  • 14. glycosylation, , -, -, site
  • 14. site, , -, sialic acid, site
  • 15. glycopeptide, , -, -, glycopeptide
  • 15. glycoproteins, , glycoproteins, -, -
  • 15. prevalent, , -, -, position
  • 17. glycopeptide, , -, -, glycopeptide
  • 18. Hex, , -, a glycopeptide, glycopeptide
  • 18. glycopeptide, , -, -, glycopeptide
  • 19. glycosylation, , -, -, sites
  • 2. glycopeptides, , -, -, glycopeptides
  • 20. glycosylation, , -, -, sites
  • 3. glycopeptides, , -, -, glycopeptides
  • 3. glycosylation, , -, -, site
  • 4. glycopeptides, , -, -, glycopeptides
  • 4. glycosylation, , -, -, sites
  • 5. glycosylation, , -, -, sites
  • 6. glycosylation, , -, -, site
  • 8. glycopeptide, , -, -, glycopeptide
  • 9. glycosylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX