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C-terminal sequence analysis

Investigation into the C-terminal part of a protein is especially of interest since micro-heterogeneity frequently occurs as a post-translation modification. Hence, identification of the C-terminus and any possible truncated forms, also called “ragged ends”, is important for proving the correct structure of biopharmaceuticals.  C-terminal sequence analysis is therefore a key part of the ICH Q6B guidelines
for characterisation and confirmation of biopharmaceuticals in support of new marketing applications.

Unlike N-terminal sequencing, there is no fully reliable direct C-terminal sequencing automated instrumental technique available anymore. Confirmation of C-terminal sequence is therefore
investigated by indirect methods like by a combination of peptide mapping strategies and Edman degradation or ES-MS/MS sequencing of the C-terminal peptide, sometimes combined, if possible, with intact molecular weight analysis. Using a special affinity chromatography step, one can also selectively isolate the C-terminal peptide(s) of a protein (see also the technical / background information). Another possibility is digestion of the intact protein with certain specific carboxy-peptidase(s) followed by amino acid analysis of the released C-terminal amino acids as a function of time.  

Quite a lot of experience enables us to choose the most appropriate methods, which will vary on
according to factors such as peptide or protein size, glycosylation state and the theoretical C-terminal amino acid sequence.