OXIDATIVE SURFACE FOOTPRINTING FOR ANTI-TUMOR ANTIBODY-ANTIGEN COMPLEXES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 该项目涉及一种名为14F7的抗体，它可以识别神经节苷脂N-乙醇基GM3。这种分子在动物细胞中很常见，但除了某些肿瘤外，在人类细胞中不存在。因此，它是一种理想的肿瘤标志物。以前对Krengel组的研究已经表征了14F7 Fab的晶体结构，并通过计算机模拟预测了抗原复合体。该项目的目的是提供实验数据，以进一步表征抗体-抗原复合体。 我们将使用氧化足迹来产生实验数据，这些数据可以用来比较抗体-抗原复合体的理论结构。该项目将从纯化的单抗中生产和纯化14F7 Fab片段。该项目的这一部分将在奥斯陆的化学系进行。然后，将对14F7 Fab进行胰酶处理，并与分子生物科学系的沃尔夫冈·埃格-雅各布森博士合作，通过质谱学对胰蛋白酶多肽进行表征。一旦这些准备工作完成，这项工作将在格鲁吉亚大学继续进行。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is concerned with an antibody called 14F7, which recognizes the ganglioside N-glycolyl GM3. This molecule is very common in animal cells, but not found in human cells except on certain tumors. It is hence an ideal tumor marker. Previous studies of the Krengel group have characterized the crystal structure of the 14F7 Fab and predicted the antigen complex by computer modeling. The aim of this project is to provide experimental data to further characterize the antibody-antigen complex. We will employ oxidative footprinting to generate experimental data that may be used to compare the theoretical structure of the antibody-antigen complex. The project will start with production and purification of 14F7 Fab fragment from purified mAbs. This part of the project will take place at the Department of Chemistry in Oslo. The 14F7 Fab will then be trypsinized and the tryptic peptides characterized by mass spectrometry in collaboration with Dr. Wolfgang Egge-Jacobsen at the Department of Molecular Biosciences. Once these preparations are completed, the work will be continued at the University of Georgia.