Epitope mapping of a monoclonal antibody against human thrombin by R/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein
Epitope mapping of a monoclonal antibody against human thrombin by R/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein
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DOI：
10.1110/ps.4670102
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发表时间：
2002-06-01
期刊：
影响因子：
8
通讯作者：
Komives, EA
中科院分区：
文献类型：
--
作者：
Baerga-Ortiz, A;Hughes, CA;Komives, EA
抗人凝血酶的单抗的表位已通过氢/氢交换-MALDI质谱仪确定。抗体表位被鉴定为凝血酶的表面，在有单抗存在的情况下，与没有单抗的对照实验相比，凝血酶表面保留了氚。抗体与蛋白G小球的共价结合，以及氢交换后抗EN的有效洗脱，使我们能够在一个MALDI质谱图中分析所有可能的表位。该表位是不连续的，由两个靠近阴离子结合的exosite 1的多肽组成，很容易被识别出来。该表位与血栓调节蛋白结合位点重叠，但不完全相同，这与抑制研究一致。该抗体与人凝血酶结合，而不与鼠或牛凝血酶结合，尽管这些蛋白与人蛋白有86%的同源性，但有趣的是，表位被证明是由两个表面区组成的更具结构的区域，在这两个表面区中，三个物种之间的序列差异较大。
The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the anti-en after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite 1, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein, Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.