N-Terminal Sequencing in Protein Analysis
N-terminal sequencing, the process of determining an amino acid sequence starting from the N-terminal (amino-terminal) of a protein, uses chemical or enzymatic degradation methods to identify the sequence of amino acids in a protein or polypeptide chain. This technique is widely used in biological research. In the discovery and identification of novel proteins, N-terminal sequencing provides direct information on amino acid sequences, which assists in predicting protein function and structure. Understanding the N-terminal sequence of a protein aids in comprehending its primary structure, which further allows for inference of its three-dimensional structure and function. For example, signal peptides are typically located at the N-terminal of proteins, and sequencing this region can reveal the signal peptide's position and sequence, elucidating the protein's secretion and localization mechanisms. In recombinant protein drug production, N-terminal sequencing verifies the correctness of the expressed product, ensuring the recombinant protein's N-terminal sequence matches the designed sequence, thereby maintaining the drug's quality and efficacy. In certain diseases, the N-terminal region of proteins may undergo abnormal modifications or cleavages; N-terminal sequencing can identify these changes, offering biomarkers for disease diagnosis and treatment. For example, variations in the N-terminal of certain tumor-related proteins might be linked to tumor initiation, progression, and metastasis. The precision and reliability of N-terminal sequencing enable researchers to gain deep insights into biomolecular mechanisms, providing a solid foundation for clinical and industrial applications.
Basic Principles of N-terminal Sequencing
1. Chemical Method
The Edman degradation method exemplifies this approach. It involves the reaction of N-terminal amino acid of a protein or polypeptide with phenylisothiocyanate (PITC) under mild alkaline conditions, forming a phenylthiocarbamoyl (PTC) derivative. Subsequently, under acidic conditions, the N-terminal amino acid in the PTC-polypeptide cyclizes to form a phenylthiohydantoin (PTH)-amino acid, which is cleaved from the peptide chain. The PTH-amino acid is then identified using high-performance liquid chromatography (HPLC), allowing determination of the N-terminal amino acid sequence. Each cycle identifies one amino acid, and repetition of this process reveals the N-terminal sequence.
2. Enzymatic Method
This method employs aminopeptidase to sequentially hydrolyze amino acids from the N-terminal of a protein or polypeptide. By analyzing the hydrolysis products, the N-terminal sequence is determined. Different aminopeptidases exhibit varying hydrolytic activities for different amino acid residues, and selecting the appropriate enzyme enables specific amino acid cleavage and analysis.
3. Mass Spectrometry Method
Using technologies such as electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI), proteins or polypeptides are ionized, and in a mass spectrometer, ions are separated and detected based on their mass-to-charge ratio (m/z). Analyzing the mass spectra of N-terminal peptide fragment ions provides amino acid sequence information. Tandem mass spectrometry (MS/MS) allows further fragmentation of peptide fragments, producing a series of fragment ions that enable determination of the amino acid sequence based on mass differences.
N-terminal Sequencing Process
1. Sample Preparation
The initial step requires extraction and purification of the target protein or polypeptide from biological samples, ensuring the sample's purity and concentration are suitable for sequencing analysis. This may involve cell lysis, protein separation, and purification steps, employing methods such as gel electrophoresis, affinity chromatography, and ion exchange chromatography.
2. Sequencing Reaction
The reaction appropriate to the chosen sequencing method is carried out. For Edman degradation, the purified protein or polypeptide reacts with PITC and other reagents under specific conditions. For mass spectrometry, the sample needs ionization to facilitate analysis in a mass spectrometer.
3. Result Analysis
In Edman degradation, instruments like HPLC analyze the elution time and peak area of PTH-amino acids, with amino acid types identified through comparison with standards. In mass spectrometry, specialized data analysis software interprets the mass spectra, identifying ion peaks correlated with amino acid sequences and deducing the N-terminal sequence.
MtoZ Biolabs offers professional N-terminal sequencing services, utilizing state-of-the-art technology platforms and expert teams to deliver high-quality sequencing results. We are dedicated to assisting clients in obtaining protein N-terminal sequence information quickly and accurately, providing reliable data support for scientific research and industrial applications. By choosing MtoZ Biolabs, you are assured of superior service and technical support.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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