PMID: PMC3975653-1-3

 

    Legend: Gene, Sites

Title : Site-Specific Analysis of HDL Glycoproteins

Abstract :
  1. Glycopeptideswere assigned based on a combination of the MS/MS data and the accurateprecursor ion mass measurement
  2. The advantage of nonspecific digestionwith Pronase is the ability of the enzyme mixture to cleave the protein(s)into short peptides with typically one glycosylation site
  3. Previousstudies from our group on the analysis of nonspecific digested glycopeptideswith collision-induced dissociation (CID) experiments revealed detailedand comprehensive glycan com position al information for each glycopeptide (both N- and O-linked)
  4. Glycosidic bond cleavages(B- and Y-type ions) were the major products as well as some minorpeptide fragmentations
  5. Table 2 liststhe Pronase-digested glycopeptides from HDL-associated glycoproteinswith their glycan com position, glycosylation site , protein ID, andintensity
  6. The most abundant N-glycan (Hexose5HexNAc4Neu5Ac2) was found in multiple glycoproteins fromthe glycopeptide assignment
  7. Here, all the identified proteins fromthe HDL were confirmed in triplicate experiments using X
  8. Tandem andwere used in the glycopeptide assignment search
  9. A number of deconvolutedglycopeptide MS/MS spectra are shown as examples in Figure 3
  10. Figure 3A represents theMS/MS spectrum for a triprotonated fetuin A glycopeptide containingthis most abundant disialylated N-glycan
  11. Particularly unique to glycopeptidescontaining sialylated glycans are B-type ions corresponding to neutralmass 291 Da (Neu5Ac) and neutral mass 273 Da (Neu5Ac-H2O).
  12. Mass peaks also include those observed as 203 Da, 365 Da, and656 Da, which correspond to the neutral masses of HexNAc, (Hex + HexNAc),and (Hex + HexNAc + Neu5Ac).
  13. Because of the labile nature of sialicacid residues and their positions at the termini , the initial lossof sialic acid was commonly observed with the sialylated glycopeptides
  14. Following the sequential neutral losses of monosaccharides of Neu5Ac,hexose, and HexNAc, the CID data revealed the mass peak 832 Da correspondingto glycopeptide (APLNDT + HexNAc)
  15. The presence of (peptide + HexNAc)is considered a valuable means for validating the assignment of glycopeptideparticularly when analyzing complex protein mixtures
  16. Pronasedigestion simultanously enables site-specific analysisof both N- and O-glycans
  17. In addition to the abundant N-glycans discussedpreviously, a number of O-glycopeptides were present attached withsialylated O-glycans (Table 2)
  18. The observedO-glycans associated with the HDL glycoproteins were all sialylated,comfirming that HDL particles are highly sialylated
  19. Figure 3B,C revealed the deconvoluted MS/MS spectra fortwo diprotonated O-glycopeptides from ApoC-III and fetuin A, respectively
  20. Most O-glycopeptides analyzed in the HDL mixture contained a core1 type with two Neu5Acs
  21. Figure 4 showsthe site heterogeneity offour glycoproteins from eight of the identified HDL associated glycoproteinsas an example
  22. Fetuin A is a multifunctional circulating liver-derivedglycoprotein in serum and plasma
  23. Severalstudies have suggested that fetuin A may be critically important tocardiovascular health
  24. Both N- and O-glycans were observed at multiple glycosylation siteswith fetuin A, all of which were sialylated (Figure 4A), which is consistent with the previous glycosylation analysison the individual protein fetuin A . Themost abundant glycan (Hexose5HexNAc4Neu5Ac2) was disialylated and was found on most of the glycoproteinsexamined
  25. It was present at ASN156 and ASN176 of fetuin A . Angiotensinogen is a heterogeneous glycoprotein mainly producedby hepatocytes in plasma, which has a well-known role in blood pressureregulation in animals and humans
  26. Ithas previously been shown that the glycosylation of angiotensinogenmay play a significant role in its functional heterogeneity
  27. Results of site heterogeneity of angiotensinogenare summarized in Figure 4B
  28. Among the fourpotential N-glycosylation sites , three identified sites were occupiedwith N-glycans in this study
  29. While two sites were observed associatedwith sialylated N-glycans, site ASN304 was attached withfucosylated N-glycans
  30. Angiotensinogen was also found attached withthe most abundant sialylated glycan (Hexose5HexNAc4Neu5Ac2) at ASN170
  31. Figure 4C details the glycan associatedwith alpha-1B-glycoprotein ( A1BG )
  32. A1BG is believed to be a memberof the immunoglobulin family
  33. The monosialylatedglycan (Hexose5HexNAc4Neu5Ac1) attachedat ASN363 on A1BG
  34. The glycosylation of apolipoproteinC3 ( ApoC-III ) was analyzedby a couple of groups, showing the distribution of O-glycans
  35. The disialylated O-glycan at site Thr94 from ApoC-IIIis shown in Figure 4D
  36. ApoC-III inhibits lipoproteinlipase and hepatic lipase , thought to inhibit hepatic uptake of triglyceride-richparticles
  37. ApoC-III was also recentlyfound to be increased in HDL isolated from patients with both stablecoronary artery disease and acute coronary syndrome as well as inhemodialysis patients
  38. In general, for the firsttime the site-specific glycosylationof these proteins in HDL particles has been described
  39. Compared withprevious disscussed glycan analysis, some glycans were not observedin the glycopeptide analysis due to their low abundances as well asthe potential short peptide length from Pronase digestion
  40. However,the functions they impart on HDL through their glycosylation, andwhether alterations in their glycosylation as part of HDL particlesis either causative or diagnostic in disease, are unknown
Output (sent_index, trigger, protein, sugar, site):
  • 10. disialylated, , fetuin A, -, -
  • 10. glycopeptide, , -, -, glycopeptide
  • 13. glycopeptides, , -, -, glycopeptides
  • 13. sialylated, , -, -, glycopeptides
  • 14. glycopeptide, , -, -, glycopeptide
  • 17. O-glycopeptides, , -, -, O-glycopeptides
  • 18. glycoproteins, , HDL glycoproteins, -, -
  • 19. O-glycopeptides, , ApoC-III, -, O-glycopeptides
  • 19. O-glycopeptides, , fetuin A,, -, O-glycopeptides
  • 2. glycosylation, , -, -, site
  • 20. O-glycopeptides, , -, -, O-glycopeptides
  • 21. glycoproteins, , glycoproteins, -, -
  • 22. liver-derivedglycoprotein, , Fetuin A, -, -
  • 22. liver-derivedglycoprotein, , liver-derivedglycoprotein, -, -
  • 24. disialylated, , -, Hexose5HexNAc4Neu5Ac2, -
  • 24. found, , glycoproteinsexamined, Hexose5HexNAc4Neu5Ac2, -
  • 24. sialylated, , fetuin A,, all, -
  • 25. glycoprotein, , Angiotensinogen, -, -
  • 25. glycoprotein, , glycoprotein, -, -
  • 28. N-glycosylation, , -, -, sites
  • 3. glycopeptide, , -, -, glycopeptide
  • 30. sialylated, , -, Hexose5HexNAc4Neu5Ac2, -
  • 31. alpha-1B-glycoprotein, , A1BG, -, -
  • 31. alpha-1B-glycoprotein, , alpha-1B-glycoprotein, -, -
  • 34. glycosylation, , ApoC-III, -, -
  • 34. glycosylation, , apolipoproteinC3, -, -
  • 39. glycopeptide, , -, -, glycopeptide
  • 5. glycopeptides, , glycoproteinswith, -, glycopeptides
  • 5. glycopeptides, , glycoproteinswith, -, site
  • 5. glycoproteinswith, , -, -, position,
  • 5. glycosylation, , -, -, site
  • 6. found, , glycoproteins, Hexose5HexNAc4Neu5Ac2, -
  • 6. glycopeptide, , -, -, glycopeptide
  • 6. glycoproteins, , glycoproteins, -, -
  • 8. glycopeptide, , -, -, glycopeptide
  • 9. deconvolutedglycopeptide, , -, -, deconvolutedglycopeptide
Output(Part-Of) (sent_index, protein, site):
  • 13. -, positions
  • 13. -, residues
  • 19. ApoC-III, O-glycopeptides
  • 19. fetuin A,, O-glycopeptides
  • 25. fetuin A, ASN156 and ASN176
  • 5. HDL, site
  • 5. Pronase, glycopeptides
  • 5. glycoproteinswith, glycopeptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX