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Title: Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry.

Authors: Nemeth, J F; Hochgesang Jr, G P; Marnett, L J; Caprioli, R M; Hochensang Jr, G P

Published In Biochemistry, (2001 Mar 13)

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.

PubMed ID: 11258925 Exiting the NIEHS site

MeSH Terms: Amino Acid Sequence; Animals; Asparagine/metabolism; Cyclooxygenase 2; Genetic Vectors/biosynthesis; Genetic Vectors/chemical synthesis; Glycopeptides/analysis; Glycopeptides/metabolism; Glycosylation; Isoenzymes/biosynthesis; Isoenzymes/chemistry*; Isoenzymes/genetics; Isoenzymes/metabolism*; Mice; Molecular Sequence Data; Peptide Fragments/analysis; Peptide Fragments/metabolism; Prostaglandin-Endoperoxide Synthases/biosynthesis; Prostaglandin-Endoperoxide Synthases/chemistry*; Prostaglandin-Endoperoxide Synthases/genetics; Prostaglandin-Endoperoxide Synthases/metabolism*; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin/metabolism

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