Title : N-Glycan Microheterogeneity in
Recombinant ITIH4
Abstract :
- To achievesite-specific characterization of N- and O-glycan microheterogeneity,we performed serial proteolysis ( GluC-trypsin and trypsin-chymotrypsin )of ITIH4 , enriched glycopeptides using HILIC chromatography, and analyzedglycopeptides via LC– ESI-MS/MS before and after treatment withneuraminidase and fucosidase
- All observed glycoforms are summarizedin Table 3
- In recombinant ITIH4 , biantennarysialylated glycans were the dominant glycoforms at N81 ; triantennary,fucosylated, and high-mannose forms were also observed
- At sites N207,N517 N207,N517, and N577 , complex fucosylated, asialo-glycans were the dominantglycoforms, though some sialylated glycoforms were also observed
- At N207 , all detected glycoforms were fucosylated, and several glycopeptideswith bifucosylated N-linked glycans were detected
- We observed thegreatest microheterogeneity at N517 , with nonfucosylated, monofucosylated,and bifucosylated bi-, tri-, and tetra-antennary N-linked glycans
- At N577 both sialylated and asialo-glycoforms were observed, as wellas singly fucosylated N-linked glycans
- Analysis of detached, permethylatedN-linked glycans from recombinant ITIH4 corroborates the glycan com positions matched to glycopeptides from CID MS/MS
- The detached glycan dataalso confirmed the abundance of fucosylated glycoforms—fucosylatedN-linked glycans accounted for 89% of the relative intensity of permethylatedglycans analyzed via MALDI-TOF MS (SupplementaryFigure 1, Supporting Information)
- Double-fucosylated N-linkedglycans contributed 3% of the N-glycan relative intensity
- Fully sialylatedN-linked glycans accounted for only 13% of the signal, confirmingglycopeptide results that also demonstrated that fully sialylatedglycans are a minor component of recombinant ITIH4 N-glycoforms
- Notably,we observed the M5 N-linked glycan in MALDI-TOF spectra, representing0.1% of the relative intensity, consistent with our previous observationthat high-mannose glycopeptides are very low in abundance
- We usedoptimized methods for analysis of recombinant ITIH4 to study site-specificglycoforms in serum-derived ITIH4
Output (sent_index, trigger,
protein,
sugar,
site):
- 1. analyzedglycopeptides, , -, -, analyzedglycopeptides
- 1. glycopeptides, , -, -, glycopeptides
- 11. N-glycoforms, , ITIH4, -, -
- 11. confirmingglycopeptide, , -, -, confirmingglycopeptide
- 12. glycopeptides, , -, -, glycopeptides
- 3. glycoforms, , -, -, N81
- 4. dominantglycoforms, , -, -, N207,N517, and N577
- 4. dominantglycoforms, , -, -, sites N207,N517
- 5. fucosylated, , -, -, N207
- 8. glycopeptides, , -, -, glycopeptides
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 3. Recombinant ITIH4, -, N81
- 4. Recombinant ITIH4, -, N207,N517, and N577
- 4. Recombinant ITIH4, -, sites N207,N517
- 5. Recombinant ITIH4, -, N207