N-Terminal Protein Sequencing
N-terminal protein sequencing is a widely used method for determining the amino acid sequence at the N-terminus of a protein. By analyzing the initial amino acid residues of a protein chain, this technique reveals crucial information about protein structure and function, providing insights into biological roles, post-translational modifications, processing mechanisms, and domain architecture. It plays an essential role in proteomics, drug discovery, and biomedical research, particularly in protein structural characterization, antibody drug development, and the identification of novel proteins.
The N-terminal, representing the starting point of a protein chain, often contains characteristic sequences such as signal peptides and residues defined by the translation initiation codon. These sequences are fundamental for identifying translation start sites, understanding protein maturation and cleavage processes, and elucidating structural and functional aspects of mature proteins. N-terminal protein sequencing not only verifies correct protein expression but also identifies processing abnormalities, degradation pathways, and protein-ligand interactions.
The Edman degradation method, introduced by Pehr Edman in 1950, remains the most established approach for N-terminal protein sequencing. This method uses phenyl isothiocyanate (PITC) to selectively react with the N-terminal amino acid residue, forming a cleavable derivative. Under acidic conditions, the derivative is released from the polypeptide chain as a stable PTH-amino acid (phenylthiohydantoin-amino acid). The PTH-amino acid is subsequently separated and identified via high-performance liquid chromatography (HPLC). Repeating this cycle enables sequential determination of N-terminal amino acid residues, allowing the reconstruction of an accurate N-terminal sequence.
Workflow of N-Terminal Protein Sequencing
1. Sample Preparation
Ensure protein purity by removing contaminants and preventing degradation.
2. Chemical Modification
React N-terminal amino acids with phenyl isothiocyanate (PITC).
3. Selective Cleavag
Under acidic conditions, release PTH-amino acid derivatives from the protein.
4. Separation and Identification
Use HPLC to separate and identify each amino acid derivative.
5. Sequence Assembly
Arrange amino acids identified in each cycle to construct the N-terminal sequence.
Advantages of N-Terminal Protein Sequencing
1. High Specificity
Precisely identifies N-terminal amino acid residues.
2. High Accuracy
Reliable and reproducible results due to stable chemical processes.
3. Detailed Sequence Information
Capable of analyzing up to 30 amino acid residues.
4. Broad Applicability
Suitable for diverse protein and peptide samples.
5. Validation of Translation Sites
Confirms translation initiation sites and protein integrity.
MtoZ Biolabs offers expert N/C-terminal protein sequencing services, integrating Edman degradation with mass spectrometry to deliver accurate and reliable results tailored to the needs of research and pharmaceutical industries.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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