Title : O-Glycopeptide Microheterogeneity in
Serum-Derived ITIH4
Abstract :
- We also characterized O-glycopeptides in serum ITIH4 by CID MS/MSand detached O-glycan analyses
- 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
- 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
- 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
- There are two serine residues in this peptide (S640, S642), each representing a potential site of glycan attachment
- On the basis of CID fragmentation alone, we are unable to determineif glycosylation is restricted to a single site
- However, the com positions of serum-derived ITIH4 detached O-glycans (SupplementaryFigure 1C, Supporting Information) suggest that the peptideis glycosylated at a single site
- 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
- 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)
- 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)
- Potential O-glycosylatedresidues include S696, T701, S702, and S709
- We detected mostly simpleO-glycan structures in detached O-glycan analyses of serum-derived ITIH4 (Supplementary Figure 1, Supporting Information)
- We also detected more complex O-glycans in detached O-glycan analyses,but these represented minor components of the mixture
- 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
- 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
- This strongly supports that one of the sitesof glycosylation is S709, which is further corroborated by evidenceof glycosylation on S709 in recombinant ITIH4
- 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):