A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains.
Peptide mixtures, precolumn-derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed-phase high-performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2-5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti-azobenzenearsonate monclonal antibodies, namely 10K44-7A1 and 10K26-12A1. The complete variable region sequences of 10K44-7A1 and 10K26-12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J.D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679-7683]. Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001391:Azo Compounds / Q000276:immunology*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D007147:Immunoglobulin Light Chains / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification*; D010132:p-Azobenzenearsonate / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001391:Azo Compounds; D007147:Immunoglobulin Light Chains; D010446:Peptide Fragments; D010132:p-Azobenzenearsonate