Fiedler W, Borchers C, Macht M, Deininger S O, Przybylski M
Faculty of Chemistry, University of Konstanz, Germany.
Bioconjug Chem. 1998 Mar-Apr;9(2):236-41. doi: 10.1021/bc970148g.
A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.
本文描述了一种用于表征蛋白质抗原中构象依赖性表位结构的新方法，该方法结合了免疫复合物的差异化学修饰与质谱肽图谱分析。成熟的表位表征方法通常不适用于构象依赖性表位，而直接的结构分析方法，如免疫复合物的X射线晶体学，仅在少数情况下取得成功。我们的方法将免疫复合物的三级结构选择性化学修饰与通过质谱肽图谱对反应产物进行分子表征相结合。通过比较游离抗原和抗体结合抗原的修饰模式，可鉴定出受抗体保护而未被修饰的残基。因此，这些残基被表征为表位结构的一部分。以特征明确的鸡蛋清溶菌酶和相应的单克隆IgM型抗体作为模型系统进行研究。对精氨酸、赖氨酸和酪氨酸残基进行了特异性修饰反应，并通过质谱肽图谱确定了游离抗原和抗体结合抗原中的修饰位点。发现抗体结合的溶菌酶中的R14残基以及K13和K96残基未被修饰，这些残基通过天然蛋白质的折叠形成了一个空间相邻的表面。相比之下，其他K和R残基以及Y20和Y23在免疫复合物中未显示出明显的修饰屏蔽。这些结果提供了对天然溶菌酶分子表位表面积的估计。
