C-terminal sequence analysis – Technical / background information
Knowing the C-terminal sequence is still of great practical importance. It allows confirmation of the
correct reading frame of a nucleotide sequence for instance, and reveals post-translational processing and the exact cleavage site. Furthermore, sequence information of both the N- and C-terminus will allow the design of oligonucleotide probes to both ends of a translated region of a gene, thus creating the tools for specific isolation of a gene by PCR or by screening a DNA library.
The determination of the C-terminal amino acid(s) of proteins, however, has always been somewhat cumbersome. Previously used methods based on carboxypeptidase digestion, are known to be prone
to errors. Direct automated C-terminal sequencing is not available anymore and also had its limitations. Specific isolation of the C-terminal peptide followed by sequencing from its N-terminus is, therefore, one
of the methods of choice. It can be performed by a combination of peptide mapping strategies and
further analysis of the C-terminal peptide by Edman degradation or ES-MS(/MS).
We have a lot of experience in developing a dedicated cleavage strategy to generate the most optimal
C-terminal fragment(s) for further investigation by sequence analysis or MS techniques. In addition to selective cleavage techniques a very elegant strategy, the specific isolation of the C-terminal peptide by using a special affinity chromatography step, is also offered.
The specific isolation of the C-terminal peptide
This method is based on the use of immobilized anhydrotrypsin (or anhydrochymotrypsin) as developed
by Ishii et al. [1-3]. Anhydrotrypsin is an catalytically inert derivative of trypsin in which the active site Serine-183 has been chemically converted to a dehydroalanine. It exhibits a much higher affinity toward product-type ligands, i.e. peptides containing arginine, lysine or aminoethyl-cysteine at the C-terminus than towards substrate-type ligands, i.e. peptides containing those amino acids internally. This opens the way for affinity purification of the C-terminal peptides of proteins. Assuming that the C-terminus is not ending with Arg or Lys, one could bind all tryptic peptides to the affinity column and elute and isolate specifically the potential C-terminal peptide(s). In the opposite situation, dealing with a protein that ends with Arg or Lys, one could digest the protein with any enzyme other than trypsin-like activity, and specifically bind the C-terminal peptide to the affinity column.
Until now, we have applied this method successfully to several proteins. The success of isolating the
C-terminal part of a protein by this method partly depends on the complexity of the digest, and, therefore, also on the primary structure of the protein to be investigated and the specificity of the enzyme used for digestion. Difficulties may arise if more then one peptide is found as potential C-terminal peptide - due to, for instance, chymotryptic activity during tryptic digestion - and no other structural information i.e. a
protein or DNA sequence, is available. However, for some applications like PCR, it is not insurmountable
to try more than one oligonucleotide to amplify the gene. Another difficulty may arise if a dipeptide is formed as the C-terminal peptide, since it may not easily be detected. In those cases, supplementary investigations are necessary to identify the real C-terminal peptide.