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首页> 外文期刊>Journal of mass spectrometry: JMS >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
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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

机译：基质辅助激光解吸-电离串联质谱分析显示霍乱弧菌O1，血清型小川和BSA的O-PS末端单糖抗原中新糖复合物中的糖基化位点

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摘要

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.
机译：我们介绍了各种半抗原-牛血清白蛋白（BSA）新糖结合物的MALDI-TOF / TOF-MS分析，这些糖结合物是通过霍乱弧菌O1血清型Ogawa的O特异性多糖的带间隔子的末端单糖的方酸化学偶联获得的，到BSA。这些分析不仅可以计算具有不同半抗原-BSA比率（4.3、6.6和13.2）的半抗原-BSA新糖结合物的分子量，而且更重要的是，还可以定位半抗原和载体之间的共价键（结合位点）蛋白。糖基化位点的确定基于对BSA消化产生的肽段的MALDI-TOF / TOF-MS分析与BSA糖缀合物消化产生的相似数据的比较，然后进行MALDI-TOF / TOF-测序糖基化肽段的MS / MS。质子化分子的产物离子扫描是用装有高碰撞能池的MALDI-TOF / TOF-MS / MS串联质谱仪进行的。高能碰撞诱导解离（CID）谱图提供了由碳水化合物半抗原的断裂和与碳水化合物半抗原的片段缀合的氨基酸序列形成的产物离子。我们能够确定赖氨酸残基上的三个缀合位点（Lys235，Lys437和Lys455）。结果表明，这些赖氨酸残基具有很高的反应性，可以与赖氨酸特异性试剂结合。我们假定这些Lys残基属于那些在三维结构的表面上在空间上更易于接近的残基。 y系列产物离子的鉴定对于各种肽的测序非常有用。一系列的a和b产物离子证实了结合肽的序列。

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