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.

Diphenyltetrazole Modified Bacterial Cellulose Film: Considerations on Heterogeneous Modification and Bioconjugation

“…We chose BSA as a model protein due to the presence of a free sulfhydryl group pointed towards the outside of its structure 31 , which can react and is essential for the proposed photoclick reaction. Immobilization experiments were conducted in PBS buffer (pH 7.4) and at room temperature.…”

Carrier diversity and chemical ligations in the toolbox for designing tumor-associated carbohydrate antigens (TACAs) as synthetic vaccine candidates

“…100 BSA or tetanus toxoid under base-catalysed ligation conditions (Michael addition) afforded the required immunogenic vaccines (56,57). Even though the average hapten content was only 4-5 TF residues per BSA (colorimetric analysis), 103 the group successfully raised a mouse monoclonal antibody called JAA-F11 that was later humanized to give hJAA-F11. 104 It has to be noticed however that the TF-BSA conjugate (56) used to raise JAA-F11, once analysed by detailed mass spectrometry, showed some populations of the conjugate containing up to 8 TF/BSA which could be consistent with the above discussion concerning optimal hapten density unto a protein of the size of BSA (67 kDa).…”