Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry.
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.
Mass Spectrometry Research Center, and Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennesee 37232-6400, USA.
J F
JF
Nemeth
G P
GP
Hochgesang
L J
LJ
Marnett
R M
RM
Caprioli
G P
GP
Hochensang
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D051546:Cyclooxygenase 2; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis; D006020:Glycopeptides / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D006020:Glycopeptides; D007527:Isoenzymes; D010446:Peptide Fragments; D001216:Asparagine; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D014357:Trypsin