Identification and Characterization of Glycosylation Sites in Carcinoembryonic Antigen (Cea) by Mass Spectrometry

Swiderek, Kristine M.; Pearson, Constance S.; Shively, John E.

doi:10.1016/b978-0-12-058757-5.50019-6

Cited by 3 publications

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“…Mass spectrometry (MS) has been used as an effective method for the analysis of protein glycosylation (9)(10)(11)(12); and tandem mass spectrometry (MS/MS), in combination with electrospray ionization, has been valuable in identifying (13)(14)(15)(16), it is possible to easily identify glycopeptides. Dissociation of protonated glycopeptides in a collision-induced decomposition (CID) experiment (17)(18)(19)(20)(21)(22)) also provides structural information regarding the amino acid sequence of the peptide, the types of sugars attached, and the residue that carries the glycosyl group. This paper reports the tryptic peptide mapping of recombinant mouse COX-2 (mCOX-2) and the glycosylation analysis of the protein.…”

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confidence: 99%

“…Through the use of precursor (parent) ion scans and marker ions such as m / z 163 [protonated hexose residue (Hex)], m / z 204 [protonated N -acetylhexosamine residue (HexNAc)], or m / z 366 (HexHexNAc) ( − ), it is possible to easily identify glycopeptides. Dissociation of protonated glycopeptides in a collision-induced decomposition (CID) experiment ( − ) also provides structural information regarding the amino acid sequence of the peptide, the types of sugars attached, and the residue that carries the glycosyl group. This paper reports the tryptic peptide mapping of recombinant mouse COX-2 (mCOX-2) and the glycosylation analysis of the protein.…”

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confidence: 99%

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Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

et al. 2001

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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.

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