PMID: PMC4739677-1-7

 

    Legend: Gene, Sites

Title : Localization of the O-Glycosylation Sites

Abstract :
  1. To further characterize the identified O-glycopeptides , the corresponding O-glycosylation sites needed to be localized
  2. In a few cases the use of Proteinase K , already generated glycopeptides that exhibit only one possible O-glycosylation site , e.g.132EGPVV[HexNAc1Hex1NeuAc1]A138 and 567DLIA[HexNAc1Hex1NeuAc2]M572 from kininogen-1 or 234AP[HexNAc1Hex1NeuAc1]HPAPPGLH244 from selenoprotein P . Noteworthy, in the first example a tryptic digest would have generated a peptide with a length of 43 aa (119FVAQCQIPAEGPVVQYDCLGCVHPIQPDLEPILR161), harboring 8 potential O-glycosylation sites
  3. This clearly illustrates a benefit of the Proteinase K digest for the O-glycan site identification
  4. When the O-glycosylation sites could not be inferred directly, glycopeptides were subjected to ETD fragmentation in a separate LC-MS run (Fig. 4B)
  5. The most prominent peaks in the acquired ETD glycopeptide spectra were the unfragmented precursor ion along with charge-reduced species; minor peaks were derived from c- and z-type peptide backbone cleavages
  6. Furthermore, fragment ions indicating either the loss of 43.018 Da (C2H3O·) from the radical cationic species or 42.016 Da (C2H2O) from the even electron species [M+H]+ were consistently detected
  7. In the literature this spectral feature was attributed to the loss of an acetyl-radical from the N-acetyl group of a HexNAc (56, 57)
  8. This in turn can support the discrimination of ETD spectra derived from glycosylated and nonglycosylated species
  9. Strikingly, and in contrast to the general mode of action of ETD, also fragmentations of the glycan moiety along the intact peptide backbone were observed, leading to a complete loss of the O-glycosylated Ser/Thr side-chain
  10. Nevertheless, the resulting fragment ions enabled a verification of the glycan com position as well as the peptide mass
  11. At first, ETD generated glycopeptide spectra were searched against the human subset of the UniProtKB/Swiss- Prot database using MASCOT, under consideration of the O-glycan modification (theoretical glycan mass used as variable modification of Ser/Thr)
  12. However, this strategy failed because of the presence of intense signals in the ETD spectrum, which correspond to: (I) the precursor ion, (II) the charge reduced precursor ion, (III) acetyl radicals ions, (IV) or glycan fragment ions
  13. These ions might be erroneously interpreted as peptide derived fragment ions by the search engine, because ETD is supposed to solely produce peptide fragment ions while keeping fragile side-chain modifications, like the glycosylation, intact
  14. To overcome this, glycopeptide spectra were exported to Bruker BioTools for manual spectra annotation
  15. Here, the identified glycopeptides were built in silico, taking into account the corresponding O-glycan moieties as well as all possible O-glycosylation sites
  16. Subsequently, the resulting in silico fragment ions (c- and z-type ions) were matched to their counterparts in the measured ETD-MS2 spectra
  17. To evaluate the spectra annotation and to discern the correct O-glycosylation site , the BioTools spectra matching score along with manual inspection of the respective spectra were considered
  18. Furthermore, public repositories, namely UniProtKB and UniCarbKB, were queried with respect to known O-glycosylation sites within the peptide in question
  19. To further asses the validity of the O-glycosylation site annotation, the site occupancy was predicted using NetOGlyc—an online tool, based on machine-learning algorithms, which allows the prediction of mucin-type O-glycosylation sites (27)
  20. For 36 of 60 identified glycopeptides the quality of the corresponding ETD spectra was acceptable - in terms of signal intensity and the number of fragment ions
  21. Overall, 31 O-glycosylation sites and regions were detected, of which 23 sites could be pinpointed (Tables I and II)
  22. Strikingly, 11 previously unknown O-glycosylation sites and regions were registered, of which 8 sites could be pinpointed
  23. Generally, O-glycosylation on threonine residues was observed more frequently than on serine (16× Thr , 7× Ser )
  24. In accordance with literature, prolines were frequently found in close vicinity to the O-glycosylation site ( positions n - 1, n + 1, n + 3), e.g.267AVP[HexNAc1Hex1NeuAc1]PV272, 343VQP[HexNAc1Hex1NeuAc1]VGA349 from alpha-2-HS-glycoprotein ( 30 )
  25. In addition also prolines in position n + 2 were found occasionally, e.g.20GPVP[HexNAc1Hex1NeuAc1]PPDNI29 from alpha-1 microglycoprotein ( protein AMBP )
Output (sent_index, trigger, protein, sugar, site):
  • 1. O-glycopeptides, , -, -, O-glycopeptides
  • 1. O-glycosylation, , -, -, sites
  • 11. glycopeptide, , -, -, glycopeptide
  • 14. glycopeptide, , -, -, glycopeptide
  • 15. O-glycosylation, , -, -, sites
  • 15. account, , -, -, sites
  • 15. glycopeptides, , -, -, glycopeptides
  • 17. O-glycosylation, , -, -, site
  • 18. O-glycosylation, , -, -, sites
  • 19. O-glycosylation, , -, -, site
  • 19. O-glycosylation, , -, -, sites
  • 2. 234AP[HexNAc1Hex1NeuAc1, , Proteinase K, 234AP[HexNAc1Hex1NeuAc1, -
  • 2. O-glycosylation, , -, -, site
  • 2. O-glycosylation, , -, -, sites
  • 2. Proteinase, , Proteinase K, 567DLIA[HexNAc1Hex1NeuAc2]M572, -
  • 2. Proteinase, , Proteinase K, e.g.132EGPVV[HexNAc1Hex1NeuAc1]A138, -
  • 2. glycopeptides, , -, -, glycopeptides
  • 2. kininogen-1, , kininogen-1, 567DLIA[HexNAc1Hex1NeuAc2]M572, -
  • 2. kininogen-1, , kininogen-1, e.g.132EGPVV[HexNAc1Hex1NeuAc1]A138, -
  • 20. glycopeptides, , -, -, glycopeptides
  • 21. O-glycosylation, , -, -, sites
  • 22. O-glycosylation, , -, -, sites
  • 23. O-glycosylation, , -, -, threonine residues
  • 23. Thr, , -, -, Ser
  • 24. O-glycosylation, , -, -, positions
  • 24. O-glycosylation, , -, -, site
  • 24. alpha-2-HS-glycoprotein, , alpha-2-HS-glycoprotein, 343VQP[HexNAc1Hex1NeuAc1]VGA349, -
  • 25. microglycoprotein, , microglycoprotein, e.g.20GPVP[HexNAc1Hex1NeuAc1]PPDNI29, -
  • 25. microglycoprotein, , protein AMBP, -, -
  • 4. O-glycosylation, , -, -, sites
  • 4. glycopeptides, , -, -, glycopeptides
  • 5. glycopeptide, , -, -, glycopeptide
  • 9. O-glycosylated, , side-chain, -, -
Output(Part-Of) (sent_index, protein, site):
  • 2. Proteinase K, glycopeptides
  • 24. alpha-2-HS-glycoprotein, site
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX