Title : Results for Influenza A
Virus Hemagglutinin
Abstract :
- HA presentsa considerably greater analytical challenge than AGP
- Due to the presenceof 9 putative glycosylation sites with a wide distribution of possibleglycan com positions at each site, the number of theoretical glycopeptides (the search space) was more than 10-fold greater than that of AGP,as shown in Table 1
- In addition, the numberof glycopeptide glycoforms detected was greater, and as a result ofhigher glycoform heterogeneity, the corresponding relative abundancesof each glycopeptide precursor ion were generally lower than thoseobserved for transferrin and AGP
- In Table 2, we compared 5 different glycopeptide com positions that were identifiedby GlycReSoft in both HILIC-C18 and C18 data from HA analyses
- Outof the 5 glycopeptides assigned by intact mass, only 1, NGSYPNLSK-[5,4,1,1,0],underwent tandem MS with C18 analysis
- However, in the case of HILIC-C18,useful tandem MS data were acquired on all 5 glycopeptides , leadingto formation of not only peptide backbone ions but also backbone ionswith an attached HexNAc , facilitating identification of the glycosylationsite
- This was important in case of NGSYPNLSK-[5,4,1,1,0], where twoglycosylation sequons, NGS and NLS, were present on the same glycopeptide ;HILIC-C18 tandem MS data allowed us to identify NLS as the occupiedsequon, as shown in Supporting Information TableS-3(c) and Figure 3
- Tandem MS was alsouseful in identifying other modifications
- For example, the exactsite of deamidation could be identified in case of HA glycopeptideNVTVTHSVNLLEDSHGK(1-Deamidation)-[7,6,1,3,0]
- As shown in Table 1, measurement of glycopeptidemass did not suffice to identify the peptide and glycan com position because of the size of the search space
- Thus, observed masses consistentwith more than one glycopeptide were not uncommon
- We showed thattandem MS could be used to resolve ambiguities
- As shown in Table 2, HA precursor ion m/z 1339.1270 (5+) matched three different com positions, SWSYIAETPNSENGTCYPGYFADYEELR-[7,6,1,3,0],NGSYPNLSKSYVNNKEK (1 deamidation)-[14,8,0,3,0], and NGSYPNLSKSYVNNK(2 deamidation)-[12,13,3,0,0], within a 10 ppm mass error tolerance
- Peptide backbone and stub glycopeptide ions proved useful in assigningSWSYIAETPNSENGTCYPGYFADYEELR-[7,6,1,3,0] asthe correct com position
- In case of AGP, SVQEIQATFFYFTPNK-[7,6,0,4,0]or QNQCFYNSSYLNVQRENGTVSR-[6,6,0,2,0] also correspondedto the same precursor mass 1088.2441 (5+), and SVQEIQATFFYFTPNK-[7,6,0,4,0]was assigned as the correct com position based on tandem MS data
- Thefact that HILIC-C18-MS resulted in relative precursor ion abundancessufficient to allow selection for tandem MS and consequently makethese assignments demonstrates the value of this approach
Output (sent_index, trigger,
protein,
sugar,
site):
- 11. glycopeptide, , -, -, glycopeptide
- 14. glycopeptide, , -, -, glycopeptide
- 2. glycopeptides, , -, -, glycopeptides
- 2. glycosylation, , -, -, sites
- 3. glycoforms, , -, -, glycopeptide
- 3. glycopeptide, , -, -, glycopeptide
- 4. glycopeptide, , -, -, glycopeptide
- 5. glycopeptides, , -, -, glycopeptides
- 6. glycopeptides, , -, -, glycopeptides
- 7. glycopeptide, , -, -, glycopeptide
- 7. twoglycosylation, , -, -, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):