N- and C-Terminal Sequencing of Proteins
N- and C-terminal sequencing of proteins refers to the determination of the amino acid sequences at both termini of a protein chain. These sequences critically influence the protein’s function, cellular localization, and post-translational modifications. Understanding a protein’s sequence is foundational for elucidating its structure and function. In functional studies, knowledge of the N- and C-terminal sequencing of proteins provides insight into cellular localization signals and interaction sites with other molecules. Furthermore, in drug development, detailed sequence information facilitates the design of more effective inhibitors or agonists targeting specific proteins. In synthetic biology, this sequence information is vital for engineering bioengineered proteins. With advances in proteomics, N- and C-terminal sequencing of proteins underpins comprehensive studies of protein function and opens new avenues in disease diagnosis and therapy.
Methods for N- and C-Terminal Sequencing of Proteins
1. Edman Degradation
Edman degradation sequentially removes and identifies N-terminal amino acids, gradually revealing the N-terminal sequence. This method requires high sample purity and is not applicable for C-terminal sequences. Under mild alkaline conditions, the α-amino group of the N-terminal reacts with phenyl isothiocyanate (PITC) to form a phenylthiocarbamoyl (PTC) protein. Acid conditions then induce cyclization of the N-terminal residue to phenylthiohydantoin (PTH)-amino acid, which can be cleaved and identified via high-performance liquid chromatography (HPLC). This iterative process allows for the determination of the N-terminal sequence in proteins with unblocked N-termini, and is utilized for sequence analysis of novel proteins and post-translational modification studies.
2. Mass Spectrometry
Mass spectrometry (MS) provides rapid, accurate determination of N- and C-terminal sequences by analyzing proteins and peptide fragments. Ionized protein samples are separated and detected based on their mass-to-charge ratio (m/z). For N-terminal sequencing, analysis of the intact protein or enzymatic fragments via software aids in sequence determination. Techniques such as electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization (MALDI-TOF MS) determine protein molecular weights, while tandem MS (MS/MS) generates fragment ions to infer the N-terminal sequence. MS techniques offer high sensitivity and resolution, crucial for analyzing trace proteins in complex samples during proteomics research.
3. Chemical Degradation
Chemical degradation employs specific reagents to decompose proteins for N-terminal or C-terminal sequencing. Carboxypeptidases hydrolyze amino acids from the C-terminal. Using carboxypeptidases with varying specificities, such as types A, B, and Y, the sequence can be deduced from the order and types of released amino acids. Kinetic monitoring of hydrolysis products over time reveals the C-terminal sequence. While suitable for most proteins, the method's efficacy may be compromised by residues like proline at the C-terminus.
Technical Procedures for N- and C-Terminal Sequencing of Proteins
1. Sample Preparation
Purity and concentration of protein samples are essential for successful sequencing, necessitating purification to eliminate impurities.
2. Enzymatic Digestion and Fragmentation
Proteins are enzymatically digested to facilitate analysis. Selecting appropriate enzymes and conditions enhances efficiency and accuracy.
3. Data Analysis
Post-sequencing, data interpretation is critical. Given the complexity of large data sets, specialized software ensures sequence accuracy.
MtoZ Biolabs offers extensive expertise in N- and C-terminal sequencing of proteins, providing high-quality analytical services for research institutions and biopharmaceutical companies. Leveraging advanced MS technologies, we ensure accuracy and reliability, delivering personalized solutions to support research endeavors effectively.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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