PMID: PMC4184449-1-6

 

    Legend: Gene, Sites

Title : Results for Influenza A Virus Hemagglutinin

Abstract :
  1. HA presentsa considerably greater analytical challenge than AGP
  2. 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
  3. 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
  4. In Table 2, we compared 5 different glycopeptide com positions that were identifiedby GlycReSoft in both HILIC-C18 and C18 data from HA analyses
  5. Outof the 5 glycopeptides assigned by intact mass, only 1, NGSYPNLSK-[5,4,1,1,0],underwent tandem MS with C18 analysis
  6. 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
  7. 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
  8. Tandem MS was alsouseful in identifying other modifications
  9. For example, the exactsite of deamidation could be identified in case of HA glycopeptideNVTVTHSVNLLEDSHGK(1-Deamidation)-[7,6,1,3,0]
  10. 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
  11. Thus, observed masses consistentwith more than one glycopeptide were not uncommon
  12. We showed thattandem MS could be used to resolve ambiguities
  13. 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
  14. Peptide backbone and stub glycopeptide ions proved useful in assigningSWSYIAETPNSENGTCYPGYFADYEELR-[7,6,1,3,0] asthe correct com position
  15. 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
  16. 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):

 

 

Protein NCBI ID SENTENCE INDEX