Still N-terminal Edman sequencing has advantages for protein analysis that cannot readily be obtained by other analysis methods. Creative Proteomics provides N-terminal sequence analysis by both Edman and Mass spectrometry of therapeutic proteins, monoclonal antibodies and protein vaccines.
Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N-terminal end. Automated N‐terminal sequence analysis involves a series of chemical reactions that derivatize and remove one amino acid at a time from the N‐terminus of purified peptides or intact proteins. At least several picomoles of a purified protein or 10 to 20 pmol of a purified peptide is required to obtain useful sequence information, and an unmodified α-amino group is required at the N-terminal end of the molecule to undergo this cyclic process.
In N-terminal analysis, after modification with phenylisothiocyanate (PITC), the derivatized terminal amino acid is removed by acid cleavage as its phenylthiohydantoin (PTH) derivative and a new α-amino group on the next amino acid is now available to react with PITC. The series of sequencer reactions results in identification of the N-terminal amino acid present on the peptide or protein at the beginning of that cycle. If each step were 100% efficient, it would be possible to sequence an entire protein in a single sequencer run, while in practice multiple factors limit the amount of sequence information that can be obtained. With current technology, it is fairly routine to obtain at least 20 to 25 residues of sequence from the N-terminus of the proteins and peptides.
Currently, the most common applications for protein sequence analysis are as follows. Verification of the N-terminal boundary of recombinant proteins, particularly proteins larger than 40–80 kDa when highly accurate masses cannot be obtained by ESI-MS. Determining the N-terminal boundary of protease-resistant domains, particularly when the protein or domain is greater than 40–80 kDa or when the domain of interest cannot be readily purified. Identifying proteins isolated from species where most of the genome has not yet been sequenced. In the increasingly rare cases where the target protein is not in available databases, the peptide sequence may be used either to design oligonucleotide probes or to confirm putative cDNA clones. Besides, sequence analysis of proteins can be used to identify modified residues or crosslinked sites in proteins that prove to be refractory to analysis by mass spectrometry. n-terminal sequencing service
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