N-Terminal Sequencing via Edman Degradation is valuable as an analysis tool for the sequencing / determination of the first 25-35 residues (from the N-terminus) of a protein or peptide. While sequencing farther is possible, success is dependent on amount, purity, and amino acid composition of the sample. Blue Stream Laboratories conducts automated N-Terminal Sequencing using > 10 pmols of relatively pure analyte. Blue Stream uses an automated Edman degradation method.
Contaminants of greater than 10% may decrease the success of obtaining an unambiguous sequence. There are also concerns regarding the presence of certain amino acids such as proline that can result in incomplete hydrolysis. This, in turn, causes residual carry-over into subsequent residues. However, this effect can be minimized if the location of each proline is known, which allows for cleavage-cycle program modification (on the sequencer) in order to improve on the hydrolysis. In addition, cysteine is not detected by this method, unless appropriately alkylated.
The heavy chains of many immunoglobulins are "blocked" and, therefore, cannot be sequenced by means of the Edman degradation reaction. Blue Stream has several sample preparation / treatment methods which may be employed to un-block the N-Terminus.
N-terminal Sequencing may be employed for:
- Identity determination of the product or impurity
- Evaluation of potential truncation of the N-Terminus
- Evaluation of product purity / degradation (as evidenced by multiple N-Termini)
- Proteomics support for protein / peptide identification
- Peptide Mapping support to determine amino acid sequence changes in selected peptide(s)
N-Terminal Sequencing is offered at Blue Stream as a cGMP release assay, as well as for research support. Our N-Terminal Sequencing capacity allows us to run up to 8 samples concurrently, with quick turnaround times.
