Can Edman Sequencing be Performed in Complex Mixtures?
The Edman sequencing method is widely used to determine the primary structure of proteins, specifically the unique sequence of amino acids in the protein chain. The Shimadzu Protein N-terminal Sequencer (PPSQ) is one such device employed for this purpose. Each protein has a distinct and defined amino acid sequence, and Edman sequencing plays a key role in decoding this sequence. This technique is crucial for protein characterization and is often the first step in understanding a protein’s functional properties.
Edman degradation, developed and automated by P. Edman in the 1960s, involves three main steps: coupling, cyclization and cleavage, and conversion, followed by chromatographic analysis. The chromatographic retention time is used to identify each amino acid. The amide bonds in the remaining peptide chain remain unaffected, allowing the next cycle of degradation to occur. Each cycle identifies one amino acid, and after all cycles, the entire amino acid sequence of the protein can be determined.
To ensure accurate amino acid sequencing, the sample entering the N-terminal sequencer must be pure. But can Edman sequencing be performed on a complex mixture? The answer is yes. The target protein can be isolated using the following methods before Edman sequencing.
1. For mixtures of proteins and small molecules, a 10KD ultrafiltration membrane can be used to centrifuge out small molecules, retaining proteins larger than 10KD. If the protein consists of smaller peptides, a C18 membrane can be used to remove the small molecules, resulting in purified protein suitable for Edman sequencing.
2. For mixtures of multiple proteins, SDS-PAGE can be used to separate proteins based on size. The proteins can then be transferred to a PVDF membrane using semi-dry transfer. After staining with Coomassie Brilliant Blue or R250, the target protein bands are selected for Edman sequencing. This method is applicable to proteins with molecular weights in the range of 10-100 kDa.
3. For mixtures of proteins or peptides with similar molecular weights that cannot be clearly separated by SDS-PAGE, reverse-phase high-performance liquid chromatography (HPLC) can be used to separate the proteins based on their elution times. Samples corresponding to specific elution times are collected and, after desalting and concentration, can be analyzed by Edman sequencing. For example, insulin can first be reduced to break the disulfide bonds between its A and B chains. The HPLC will yield two distinct peaks, which can then be separately collected to obtain the A and B chains of insulin.
Edman sequencing can indeed be performed in a complex mixture, provided that the N-terminal of the protein is not blocked or chemically modified and that the protein's size is appropriate for Edman sequencing. For proteins that are unsuitable for Edman sequencing, alternative methods, such as mass spectrometry, can be used for complementary analysis.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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