Determination of glycation sites by tandem mass spectrometry in a synthetic lactose‐bovine serum albumin conjugate, a vaccine model prepared by dialkyl squarate chemistry

Abstract

RATIONALE Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. METHOD Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. RESULTS We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. CONCLUSIONS It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially.

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

“…For the determination of the average number of carbohydrate‐spacer moieties linked to BSA (TF antigen:BSA), the two glycoconjugates were analyzed by matrix assisted laser desorption/ionization‐mass spectrometry (Fig. a and b and Table ) . The MALDI‐TOF‐MS was characterized by the formation of a protonated molecular ions [M + H] + at m/z 67 599 and 70 905 and their TF–BSA ratios were calculated to be individually 2:1 and 8:1.…”

“…BSA is one of the most widely studied proteins used as a carrier protein for the synthesis of neoglycoconjugates. [27,[37][38][39][40][41] BSA contains 35 cysteine residues linked by S-S disulphide bonds among which, however, there is only a single buried free cysteine residue (Cys34) [42] that has been systematically used for other conjugation. [43,44] It is known that the 35 cysteine residues of BSA form 17 disulphide bonds at these positions (1) (17) 581-590 .…”

“…2a and 2b and Table 1). [38][39][40][41] The MALDI-TOF-MS was characterized by the formation of a protonated molecular ions [M + H] + at m/z 67 599 and 70 905 and their TF-BSA ratios were calculated to be individually 2:1 and 8:1.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Effect of sialylation pattern on immune response (Cai et al, 2012) 6-Sulfated GlcNAc mimics BSA R-TOF-TOF (sinapinic) 6-SO 3 -GlcNAc-containing sugar shown to mimic the sulfated cruzipain epitope that plays a central role in immune recognition Sulfo-Lewis A and (GlcNAc) 3 Ovalbumin MALDI For targeting mannose receptor (Singh et al, 2011c) (Jahouh, et al, 2012) obtain information on the nature of the monosaccharide building blocks of the oligosaccharides. Sialylated and sulfated glycans remain a problem, although sialylated glycans can be handled after suitable derivatization, particularly by permethylation or simply by methyl ester or methyl amide formation.…”

Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

“…For smaller, commonly used carrier proteins (<100 kDa) such as ovalbumin, BSA, and thyroglobulin (TG), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) is an accurate and reliable analytical method. 5 , 7 − 9 …”