Title : Localization of the O-Glycosylation Sites
Abstract :
- To further characterize the identified O-glycopeptides , the corresponding O-glycosylation sites needed to be localized
- 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
- This clearly illustrates a benefit of the Proteinase K digest for the O-glycan site identification
- When the O-glycosylation sites could not be inferred directly, glycopeptides were subjected to ETD fragmentation in a separate LC-MS run (Fig. 4B)
- 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
- 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
- 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)
- This in turn can support the discrimination of ETD spectra derived from glycosylated and nonglycosylated species
- 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
- Nevertheless, the resulting fragment ions enabled a verification of the glycan com position as well as the peptide mass
- 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)
- 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
- 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
- To overcome this, glycopeptide spectra were exported to Bruker BioTools for manual spectra annotation
- Here, the identified glycopeptides were built in silico, taking into account the corresponding O-glycan moieties as well as all possible O-glycosylation sites
- Subsequently, the resulting in silico fragment ions (c- and z-type ions) were matched to their counterparts in the measured ETD-MS2 spectra
- 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
- Furthermore, public repositories, namely UniProtKB and UniCarbKB, were queried with respect to known O-glycosylation sites within the peptide in question
- 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)
- 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
- Overall, 31 O-glycosylation sites and regions were detected, of which 23 sites could be pinpointed (Tables I and II)
- Strikingly, 11 previously unknown O-glycosylation sites and regions were registered, of which 8 sites could be pinpointed
- Generally, O-glycosylation on threonine residues was observed more frequently than on serine (16× Thr , 7× Ser )
- 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 )
- 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):