Takahashi N, Takahashi Y, Putnam F W
J Chromatogr. 1983 Aug 26;266:511-22. doi: 10.1016/s0021-9673(01)90922-7.
Rapid high-performance liquid chromatographic procedure forms part of the strategy for determining the amino acid sequence of proteins. It was applied to the light chain of an immunoglobulin D, and the result, together with previous data on the sequence of the heavy chain, established the complete covalent structure of a human IgD immunoglobulin. We aimed for rapid purification of both small and large peptides. The two-step chromatography system ("two-dimensional chromatography") consists of a combination of macroreticular cation-exchange chromatography as the first dimension and reversed-phase chromatography as the second. This procedure was used for the systematic separation of the tryptic peptides of the light chain. In order to obtain overlapping of the tryptic peptides, chemical modifications of the light chain and reversed-phase chromatography were found to be the most reliable path toward the sequence analysis and greatly accelerated the determination of the light chain.
快速高效液相色谱法是确定蛋白质氨基酸序列策略的一部分。它被应用于免疫球蛋白D的轻链，其结果与先前关于重链序列的数据一起，确定了人IgD免疫球蛋白的完整共价结构。我们的目标是快速纯化大小肽段。两步色谱系统（“二维色谱”）由作为第一维的大孔阳离子交换色谱和作为第二维的反相色谱组合而成。该方法用于轻链胰蛋白酶肽段的系统分离。为了获得胰蛋白酶肽段的重叠，发现轻链的化学修饰和反相色谱是进行序列分析最可靠的途径，并大大加快了轻链的测定。
