Two-dimensional high-performance liquid chromatography and chemical modification in the strategy of sequence analysis. Complete amino acid sequence of the lambda light chain of human immunoglobulin D

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.