title: Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry.
abstract: We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.
authors.affiliation: Department of Chemistry, Memorial University of Newfoundland, Saint John's, NL, Canada.
authors.forename: Farid
authors.initials: F
authors.lastname: Jahouh
authors.forename: Rina
authors.initials: R
authors.lastname: Saksena
authors.forename: Donatella
authors.initials: D
authors.lastname: Aiello
authors.forename: Anna
authors.initials: A
authors.lastname: Napoli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Sindona
authors.forename: Pavol
authors.initials: P
authors.lastname: Kováč
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Banoub
mesh_terms: D000818:Animals; D002417:Cattle; D006001:Glycoconjugates / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006241:Haptens / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D019081:O Antigens / Q000737:chemistry* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D041241:Vibrio cholerae O1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006001:Glycoconjugates; D006241:Haptens; D019081:O Antigens; D012710:Serum Albumin, Bovine; D008239:Lysine