The Edman degradation process is a vital technique in protein sequencing, allowing for the identification of amino acids in a peptide chain. This method can be automated using an advanced instrument known as a sequenator, which efficiently mixes reagents such as phenylisothiocyanate (PITC), trifluoroacetic acid, and aqueous acid. The sequenator is highly sensitive, capable of detecting as little as 1 nanogram or 10 picomoles of an amino acid, making it an invaluable tool in biochemical analysis.
Once the Edman degradation process generates phenylthiohydantoin (PTH) amino acids, these products can be analyzed using high-performance liquid chromatography (HPLC). HPLC is essential for identifying unknown PTH amino acids by comparing their elution positions to those of known amino acid controls. This comparative analysis allows for accurate identification based on the retention time of the amino acids as they elute from the HPLC column.
It is important to note that mass spectrometry can struggle to differentiate between isomeric amino acids, such as leucine and isoleucine, which share the same mass and chemical formula. In contrast, Edman degradation provides a clearer advantage in sequencing due to its ability to resolve these ambiguities. The HPLC chromatogram reveals the amino acid sequence from the N-terminal to the C-terminal end of the peptide, with the sequence displayed from left to right. Each peak on the chromatogram corresponds to a specific amino acid, allowing for straightforward interpretation of the sequence.
For example, in an HPLC chromatogram, the first peak on the left may represent aspartic acid, followed by glutamic acid, asparagine, and so forth, continuing through the sequence. This contrasts with mass spectrometry, where the sequence is read from right to left. Understanding these differences is crucial for accurately interpreting protein sequences and utilizing the appropriate analytical techniques in biochemical research.
