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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.

SUBMITTER: Jahouh F 

PROVIDER: S-EPMC3417241 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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

Jahouh Farid F   Hou Shu-jie SJ   Kováč Pavol P   Banoub Joseph H JH  

Rapid communications in mass spectrometry : RCM 20120401 7


<h4>Rationale</h4>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.<h4>Method</h4>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 reactio  ...[more]

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