Amino Acids, Peptides and Proteins - Devenyi T., Gergely J. 1976

Medium- and high-voltage electrophoresis methods
Diagonal electrophoresis method
Isolation of the C-terminal peptide from tryptic protein hydrolysates

Due to the Specificity of tryptic Cleavage, the resulting Peptides possess a C-terminal Lysine or Arginine, except for the C-terminal peptide of the protein itself (unless, of course, it also terminates in lysine or arginine). Carboxypeptidase B specifically cleaves C-terminal basic Amino Acids, resulting in a loss of charge in the terminal peptides. If a peptide lacks lysine or arginine at its C-terminus—meaning it is indeed the true C-terminal peptide of the protein—it will not lose its charge upon carboxypeptidase Treatment.

The protein under study is digested with Trypsin, and the hydrolysate is subjected to paper Electrophoresis. Guided by a control strip, a strip is cut out for analytical diagonal electrophoresis and gently sprayed with carboxypeptidase B dissolved in 0.1% ammonium bicarbonate. The sprayed strip is placed in a humidified desiccator at 37 °C for 1 h. Following incubation, the strip is dried and subjected to electrophoresis again, perpendicular to the initial direction.

All Peptides with a C-terminal lysine or arginine migrate off the diagonal, leaving only the C-terminal peptide of the protein under study in the diagonal position.