Title : Characterization of the glycosylation
sites in
cyclooxygenase-2 using mass spectrometry
Abstract :
- Cyclooxygenase is involved in the biosynthesis and function of prostaglandins
- It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2
- This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa
- Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars
- The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites
- The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides
- The peptides differed by 162 Da which further suggested high-mannose sugars
- Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580
- It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides
- Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycosylation, , -, -, sites
- 10. non-glycosylated, , -, -, peptide
- 2. glycoprotein, , glycoprotein, -, -
- 3. glycosylation, , COX-2, -, site
- 6. glycopeptides, , -, -, glycopeptides
- 8. glycosylation, , -, -, Asn 53 and Asn 130 sites
- 8. peptides, , -, -, Asn 396-Arg 414
- 8. peptides, , -, -, Asn 580
- 8. peptides, , -, -, Thr 576-Arg 587
- 9. glycopeptides, , -, -, glycopeptides
- 9. glycosylated, , -, -, Asn 396 site
Output(Part-Of) (sent_index,
protein,
site):
- 0. cyclooxygenase-2, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 8. cyclooxygenase-2, -, Asn 396-Arg 414
- 8. cyclooxygenase-2, -, Asn 53 and Asn 130 sites
- 8. cyclooxygenase-2, -, Asn 580
- 8. cyclooxygenase-2, -, Thr 576-Arg 587
- 9. cyclooxygenase-2, -, Asn 396 site