Practical Protein Chemistry - A. Darbre 1989

Determination of C-terminal amino acid sequence
Isolation and identification of C-terminal peptides
Methods of ion-exchange chromatography

Several Methods have been proposed for the isolation and identification of the C-terminal regions of Proteins AND Peptides. All of these techniques ensure the selective Separation of the C-terminal peptide from all other peptides using HPLC, Ion-exchange Chromatography, two-way high-voltage Electrophoresis, or specific binding to solid-phase Supports.

It is unlikely that the methods described below will find widespread application; they are rather intended for specific, exceptional cases. An attempt was made to isolate the C-terminal peptide of ß-galactosidase by tryptic Digestion of the maleylated protein (to restrict Cleavage exclusively to Arg residues), followed by maleylation of the peptide mixture and passing it through a Dowex-50 Column at pH 3–4 [100]. The underlying assumption was that all peptides, except for the C-terminal one, would carry a positive charge (due to the presence of Arg residues) and thus be retained on the column (binding to the ion-exchange resin), while the uncharged C-terminal peptide alone would pass through unhindered. In practice, this approach proved unsuccessful because the C-terminal peptide of ß-galactosidase is insoluble at acidic pH values. Furthermore, this method is inapplicable to proteins that have a C-terminal Arg or contain an Arg-Pro sequence or a His residue within the C-terminal peptide.

The C-terminal Peptides of the Insulin A-chain, Lysozyme, cytochrome c, and Trypsin were isolated after blocking all free carboxyl groups with glycinamide using a Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDAC) followed by proteolytic cleavage with trypsin [33]. All tryptic peptides, with the exception of the blocked C-terminal one, contained free carboxyl groups and bound to the AG-1-X2 anion-exchange resin (Bio-Rad). Prior to chromatography, the hydrolysate was treated with carboxypeptidase B because positively charged Arg-containing peptides would otherwise fail to bind to the resin and would elute alongside the C-terminal peptide. Free Arg was separated from the C-terminal peptide by Gel filtration or cation-exchange chromatography. Although this multi-step Procedure appears lengthy, it is recommended as a straightforward and relatively rapid approach.