Protein Chemistry
Protein Sequencing: Technology
Edman Degradation
Edman degradation relies on both the high chemical reactivity of a protein's terminal amino acid and the ability to remove the selectively derived amino acid from the protein while leaving the rest of the peptide chain intact. Each cycle of the degradation occurs at the newly formed amino-terminal amino acid left by the preceding degradation. Repetitive cycles thus provide sequential removal of the amino acids from the primary structure of the peptide chain. A cycle is divided into three steps: coupling, cleavage, and conversion. See figure below.
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Protein Coupling |
The sequencing process is not completed by Edman degradation alone. Once the amino acids are removed from the sample, they must be analyzed to determine their identity. The amino acid derived from each cycle is identified by comparison to the retention time on reverse phase high performance liquid chromatography (HPLC/cLC) to the retention time of the PTH amino acid standards. Amino acids are separated on a C18 reverse phase column using acidic tetrahydrofuran with an increasing acetonitrile/propanol gradient. Sensitivity of the HPLC/cLC system is the main component in determining the sensitivity of the sequencing system.
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