N-Terminal Sequencing Method
The N-Terminal sequencing method is primarily employed to ascertain the amino acid sequence at the N-terminus of proteins or peptides. This method encompasses techniques such as Edman degradation and mass spectrometry analysis. First introduced in the 1950s, the Edman degradation technique determines sequences by systematically removing and identifying the N-terminal amino acids of a peptide chain. Noted for its high specificity and accuracy, it is particularly effective for analyzing short-chain peptides. Conversely, mass spectrometry offers high sensitivity and throughput, enabling rapid analysis of complex protein mixtures, establishing it as a predominant method for N-Terminal sequencing today. In the biopharmaceutical industry, the N-Terminal sequencing method is pivotal for verifying the concordance of the N-terminal sequence of recombinant protein drugs with anticipated designs, ensuring drug quality and safety. This application assists in the quality control of biopharmaceutical products, effectively detecting potential N-terminal modifications or truncations during protein processing. In microbiology research, N-Terminal sequencing aids in identifying secreted or surface-displayed proteins, offering insights into the physiological functions and adaptive mechanisms of microorganisms. Analyzing these proteins' N-terminal sequences can infer the presence of signal peptides, facilitating predictions of protein localization and function. In plant sciences, the N-Terminal sequencing method is utilized to examine novel proteins synthesized in response to environmental stimuli. By contrasting the N-terminal sequences of experimental and control groups, researchers can identify key proteins involved in environmental response, providing a molecular foundation for plant improvement and breeding. MtoZ Biolabs, leveraging extensive expertise and a cutting-edge technical platform, delivers professional N-Terminal sequencing services, empowering clients to achieve significant advancements in protein research.
Edman Degradation Method
1. Technical Process
The Edman degradation method involves sequential cleavage and labeling of the N-terminal amino acids of a peptide chain, followed by identification of these amino acids through high-performance liquid chromatography (HPLC). The sample's peptide chain reacts with phenylisothiocyanate (PITC) to form a cleavable cyclic derivative. The N-terminal amino acid is then removed via acidolysis, and after a series of elution and separation steps, the amino acid type is identified using HPLC.
2. Precautions
Ensuring the sample's purity and integrity is crucial, as impurities can interfere with the reaction. Moreover, strict control over experimental conditions such as temperature and pH is necessary to maintain reaction efficiency and accuracy.
3. Common Problems
The Edman degradation method is unsuitable for peptides with blocked N-termini, and for long-chain peptides, the reaction efficiency diminishes over time, complicating the accurate determination of subsequent amino acids.
Mass Spectrometry Analysis
1. Technical Process
Mass spectrometry analysis, often paired with liquid chromatography (LC-MS), allows direct analysis of protein samples. The sample undergoes ionization, producing charged peptide fragments. These fragments are accelerated in an electric field, separated by a mass analyzer, and detected, generating a mass spectrum for analysis.
2. Precautions
The quality of sample preparation is critical to the accuracy of mass spectrometry results. Complex samples might require pre-separation and purification. Furthermore, careful calibration and parameter setting of the mass spectrometer are essential.
3. Common Problems
Ion suppression effects in complex mixtures can reduce the detection sensitivity for certain components. Additionally, data analysis and interpretation demand specialized knowledge and experience.
MtoZ Biolabs offers precise and reliable N-terminal sequencing services, focusing on accurate protein sequence analysis. Our team, with significant expertise and specialized knowledge, is committed to delivering efficient technical support to our clients. Through our comprehensive services, clients can swiftly acquire the necessary protein sequence information, facilitating significant breakthroughs in both research and application domains. We invite collaboration to explore the vast potential of protein research further.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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