PMID: PMC4084840-1-8

 

    Legend: Gene, Sites

Title : O-Glycopeptide Microheterogeneity in Serum-Derived ITIH4

Abstract :
  1. We also characterized O-glycopeptides in serum ITIH4 by CID MS/MSand detached O-glycan analyses
  2. Glycopeptide IPKPEASFSPR+ HexNAc1Hex1NeuAc1 ,and the doubly sialylated [IPKPEASFSPR + HexNAc1Hex1NeuAc2 + 3H]3+ format m/z 726.01 with one missed GluCcleavage (at glutamic acid ) were observed in both serum and recombinant ITIH4
  3. An alternately cleaved glycopeptide , ASFSPR, consistent withcleavage by GluC at the glutamic acid residue , is observed with thesame glycan com positions, including [ASFSPR + HexNAc1NeuAc1 + 2H]2+m/z 579.78,[ASFSPR + HexNAc2Hex2 + 2H]2+ at m/z 653.66, [ASFSPR + HexNAc1Hex1NeuAc1 + 2H]2+ at m/z 660.79, and [ASFSPR + HexNAc1Hex1NeuAc2 + 2H]2+ at m/z 806.36, as summarized inTable 3B
  4. A CID fragmentation spectrum of[ASFSPR + HexNAc1Hex1NeuAc2 + 2H]2+ (m/z 806.36)in Figure 3B shows multiple glycopeptide Yions with the intact peptide and a partially fragmented glycan attachedto the peptide backbone
  5. There are two serine residues in this peptide (S640, S642), each representing a potential site of glycan attachment
  6. On the basis of CID fragmentation alone, we are unable to determineif glycosylation is restricted to a single site
  7. However, the com positions of serum-derived ITIH4 detached O-glycans (SupplementaryFigure 1C, Supporting Information) suggest that the peptideis glycosylated at a single site
  8. In ITIH4 purified from pooledhuman serum we detect glycoforms of both LAILPASAPPATSNPDPAVSR and LAILPASATPATSNPDPAVSR, indicating that both the canonical ITIH4 sequence and the P698 to T variant are present in the sample
  9. The glycan com positions observed on LAILPASAPPATSNPDPAVSR in serum-derived ITIH4 include HexNAc2Hex2NeuAc2, HexNAc2Hex2NeuAc3, HexNAc3Hex3NeuAc3, HexNAc3Hex3NeuAc4, and HexNAc4Hex4NeuAc4 and are similar to those observed on the T698 variant peptide (Table 3)
  10. Despite thedifference in sequence , the most common glycoforms (HexNAc2Hex2NeuAc2 and HexNAc3Hex3NeuAc3) associated with bothpeptides have similar com positions and relative intensities in serum ITIH4 (Table 3)
  11. Potential O-glycosylatedresidues include S696, T701, S702, and S709
  12. We detected mostly simpleO-glycan structures in detached O-glycan analyses of serum-derived ITIH4 (Supplementary Figure 1, Supporting Information)
  13. We also detected more complex O-glycans in detached O-glycan analyses,but these represented minor components of the mixture
  14. The most likelyexplanation for these observations is that the observed glycan com positions, including HexNAc3Hex3NeuAc3, and HexNAc3Hex3NeuAc4, represent the sum of multiple smaller O-glycans divided among threeglycosylated residues in the peptide
  15. In the glycopeptide CID MS/MSspectrum of [LAILPASAPPATSNPDPAVSR + HexNAc3Hex3NeuAc3 + 4H]4+ (m/z 1004.2) from serum-derived ITIH4 shown in Figure 3A the y5 ion (m/z 529.31, 1+) as well as the y5 ion with HexNAc(m/z 732. 39, 1 +) andHexNAc-Hex (m/z 894.44, 1+) are clearly present
  16. This strongly supports that one of the sitesof glycosylation is S709, which is further corroborated by evidenceof glycosylation on S709 in recombinant ITIH4
  17. We also observe theseions (y5 at m/z 529.31, 1+; y5 \+HexNAc at m/z 732. 39, 1 +; and y5 + HexNAc-Hex at m/z 894.44, 1+) in the P698 to T variantglycopeptides in serum and recombinant ITIH4 , indicating that in allcases S709 is glycosylated
Output (sent_index, trigger, protein, sugar, site):
  • 1. O-glycopeptides, , -, -, O-glycopeptides
  • 14. threeglycosylated, , -, -, residues in
  • 15. glycopeptide, , -, -, glycopeptide
  • 17. variantglycopeptides, , -, -, variantglycopeptides
  • 2. observed, , ITIH4, Glycopeptide IPKPEASFSPR+ HexNAc1Hex1NeuAc1, -
  • 3. glycopeptide, , -, -, glycopeptide
  • 4. glycopeptide, , -, -, glycopeptide
  • 7. glycosylated, , -, -, site
Output(Part-Of) (sent_index, protein, site):
  • 3. GluC, glutamic acid residue
  • 8. ITIH4, sequence
  • 9. T698, peptide
  • 9. variant, peptide
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX