PMID: PMC4084840-1-9

 

    Legend: Gene, Sites

Title : O-Glycosylation Site Determination

Abstract :

1. To further explore the sites of O-glycosylation in serum-derived ITIH4 , we used targeted electron-transfer dissociation (ETD) on ITIH4glycopeptides
2. As opposed to most of our CID analyses, ETD of glycopeptidesresults in fragmentation of the peptide backbone rather than of glycosidiclinkages
3. Peptide c- and z-ionsresulting from ETD fragmentation of glycopeptides retain unfragmentedcarbohydrate moieties, which can be used to determine the site(s)of glycosylation
4. We labeled the serum-derived ITIH4 glycopeptideswith mTRAQ reagents to boost the charge, treated them with a broad-specificityneuraminidase to remove sialic acids, and then subjected them to ETD(Supplementary Figure 4, Supporting Information)
5. On the basis of the MS2 spectrum of glycopeptide IPKPEASFSPR,S640 is O-glycosylated, while S642 is not
6. This is supported by thepresence of c7, c8, and c9 and z5, z6, and z7 ionswith HexNAc-Hex in the fragmentation spectrum, as well as the absenceof this modification on z3, z4, c5, and c6 ions
7. ETD fragmentation of the desialylated, mTRAQ-labeledLAILPASAPPATSNPDPAVSR + HexNAc2Hex2 peptide from serum ITIH4 indicates that theselected glycopeptide is glycosylated at S696 and T701, based on c- and z-ions in the MS2 spectrum (Supplementary Figure 4, Supporting Information)
8. We were unable to obtain information on the desialylated LAILPASAPPATSNPDPAVSR+ HexNAc3Hex3 glycopeptide , so we arenot able to corroborate the CID results that also indicate that S709is O-glycosylated
9. On the basis of the available information, themost likely explanation is that LAILPASAPPATSNPDPAVSRis O-glycosylated at S696, T701 and S709
10. We detect similar glycancom positions associated with peptides LAILPASATPATSNPDPAVSR and LAILPASAPPATSNPDPAVSR in serum ITIH4 , suggesting thatthe sequence variant is not glycosylated differently

Output (sent_index, trigger, protein, sugar, site):

• 1. ITIH4glycopeptides, , -, -, ITIH4glycopeptides
• 1. O-glycosylation, , ITIH4, -, sites
• 10. glycosylated, , variant, -, -
• 10. peptides, , -, -, peptides
• 3. glycopeptides, , -, -, glycopeptides
• 5. glycopeptide, , -, -, glycopeptide
• 7. desialylated, , -, -, peptide
• 7. glycopeptide, , -, -, glycopeptide
• 7. glycosylated, , -, -, glycopeptide
• 8. desialylated, , -, -, glycopeptide
• 8. glycopeptide, , -, -, glycopeptide

Output(Part-Of) (sent_index, protein, site):

• 1. ITIH4, sites
• 7. ITIH4, peptide

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX