Title : Site-Specific Analysis of
HDL Glycoproteins
Abstract :
- Glycopeptideswere assigned based on a combination of the MS/MS data and the accurateprecursor ion mass measurement
- 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
- 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)
- Glycosidic bond cleavages(B- and Y-type ions) were the major products as well as some minorpeptide fragmentations
- Table 2 liststhe Pronase-digested glycopeptides from HDL-associated glycoproteinswith their glycan com position, glycosylation site , protein ID, andintensity
- The most abundant N-glycan (Hexose5HexNAc4Neu5Ac2) was found in multiple glycoproteins fromthe glycopeptide assignment
- Here, all the identified proteins fromthe HDL were confirmed in triplicate experiments using X
- Tandem andwere used in the glycopeptide assignment search
- A number of deconvolutedglycopeptide MS/MS spectra are shown as examples in Figure 3
- Figure 3A represents theMS/MS spectrum for a triprotonated fetuin A glycopeptide containingthis most abundant disialylated N-glycan
- Particularly unique to glycopeptidescontaining sialylated glycans are B-type ions corresponding to neutralmass 291 Da (Neu5Ac) and neutral mass 273 Da (Neu5Ac-H2O).
- 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).
- 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
- 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)
- The presence of (peptide + HexNAc)is considered a valuable means for validating the assignment of glycopeptideparticularly when analyzing complex protein mixtures
- Pronasedigestion simultanously enables site-specific analysisof both N- and O-glycans
- In addition to the abundant N-glycans discussedpreviously, a number of O-glycopeptides were present attached withsialylated O-glycans (Table 2)
- The observedO-glycans associated with the HDL glycoproteins were all sialylated,comfirming that HDL particles are highly sialylated
- Figure 3B,C revealed the deconvoluted MS/MS spectra fortwo diprotonated O-glycopeptides from ApoC-III and fetuin A, respectively
- Most O-glycopeptides analyzed in the HDL mixture contained a core1 type with two Neu5Acs
- Figure 4 showsthe site heterogeneity offour glycoproteins from eight of the identified HDL associated glycoproteinsas an example
- Fetuin A is a multifunctional circulating liver-derivedglycoprotein in serum and plasma
- Severalstudies have suggested that fetuin A may be critically important tocardiovascular health
- 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
- 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
- Ithas previously been shown that the glycosylation of angiotensinogenmay play a significant role in its functional heterogeneity
- Results of site heterogeneity of angiotensinogenare summarized in Figure 4B
- Among the fourpotential N-glycosylation sites , three identified sites were occupiedwith N-glycans in this study
- While two sites were observed associatedwith sialylated N-glycans, site ASN304 was attached withfucosylated N-glycans
- Angiotensinogen was also found attached withthe most abundant sialylated glycan (Hexose5HexNAc4Neu5Ac2) at ASN170
- Figure 4C details the glycan associatedwith alpha-1B-glycoprotein ( A1BG )
- A1BG is believed to be a memberof the immunoglobulin family
- The monosialylatedglycan (Hexose5HexNAc4Neu5Ac1) attachedat ASN363 on A1BG
- The glycosylation of apolipoproteinC3 ( ApoC-III ) was analyzedby a couple of groups, showing the distribution of O-glycans
- The disialylated O-glycan at site Thr94 from ApoC-IIIis shown in Figure 4D
- ApoC-III inhibits lipoproteinlipase and hepatic lipase , thought to inhibit hepatic uptake of triglyceride-richparticles
- 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
- In general, for the firsttime the site-specific glycosylationof these proteins in HDL particles has been described
- 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
- 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):