title: Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys-34) of BSA. Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction.
abstract: We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier.
authors.affiliation: Department of Biochemistry, Memorial University of Newfoundland, St. John's Newfoundland, A1B 3X9, Canada.
authors.forename: Wael L L
authors.initials: WL
authors.lastname: Demian
authors.forename: Naresh
authors.initials: N
authors.lastname: Kottari
authors.forename: Tze Chieh
authors.initials: TC
authors.lastname: Shiao
authors.forename: Edward
authors.initials: E
authors.lastname: Randell
authors.forename: René
authors.initials: R
authors.lastname: Roy
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Banoub
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry*; D019496:Cancer Vaccines / Q000737:chemistry*; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D006001:Glycoconjugates / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D019496:Cancer Vaccines; D006001:Glycoconjugates; D012710:Serum Albumin, Bovine; C016467:Thomsen-Friedenreich antigen; D003545:Cysteine
keywords: MALDI-TOF-MS, LC-ESI-QqTOF-MS/MS; Thomsen-Friedenreich antigen; anti-cancer vaccine; glycation sites