Convenient Peptide Mapping of Immunoglobulin G2b and Differentiation between Leucine and Isoleucine Residues by Mass Spectrometry Using ^2H-Labeled Leucine

概要

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A new methodology using 2H-labeled leucine (Leu(D10)) was developed for peptide mapping of a large protein monoclonal immunoglobulin G2b (IgG2b). This method enabled easy assignment of molecular masses of digested peptides to an amino acid sequence of the original protein. It also enabled leucine and isoleucine residues to be distinguished from each other. Mouse 2H-labeled IgG2b (IgG2b(d)) was produced in the cells grown in a serum-free medium that contained Leu(D10) instead of Leu. The molecular mass of a peptide containing one leucine residue produced by the enzymatic digestion of RCM (reduced and carboxymethylated)-IgG2b(d) was 9 u larger than that of an unlabeled peptide. It was revealed that the number of leucine residues in the digested peptide was estimated easily by the mass difference between labeled and unlabeled peptides. Peptide mappings of RCM-IgG2b, RCM-IgG2b(d), and their equivalent mixture, RCM-IgG2b(mix), were obtained by digestion with lysyl-endopeptidase and consecutive MALDI-TOFMS analyses. It was easy to attribute the mass values to the peptides by the knowledge of the number of leucine residues, molecular masses of peptides and specificity of an enzyme. Leucine and isoleucine residues were easily distinguished in a protein when the protein contained 2H-labeled leucine residue.
日本質量分析学会の論文
1998-02-01