Sequencing of anti-thyroxine monoclonal antibody Fab fragment by ion trap mass spectrometry

Abstract

A comprehensive mass spectrometric strategy is described for the sequencing of anti-thyroxine monoclonal antibody Fab region (48 000 Da). After reduction and S-carboxymethylation of the Fab, the modified light chain and Fd fragment were separated and subjected to multiple proteolytic digestions. The resulting digests were characterized by on-line microbore liquid chromatography/electrospray ionization ion trap mass spectrometry. Database search against published immunoglobulins (IgGs) allowed identification of all the peptides in constant domains. The homologous framework residues in the IgGs were utilized as 'sequence maps' for the sequence determination of variable domains. S-Carboxymethylation with an isotopic-enriched moiety greatly facilitated the recognition and data elucidation of cysteinyl peptides through the unique isotopic distribution patterns specific to the modified peptides. Methylation of peptide mixtures provided additional information for the interpretation of MS/MS spectra, allowing easy differentiation of Asp/Asn and Gln/Glu pairs. This study clearly demonstrates the power of mass spectrometry for the sequencing of antibodies without knowing the corresponding DNA sequences.