C-Terminal Sequencing Based on MS: Methods and Applications
C-terminal sequencing is a technique specifically designed to determine the amino acid sequence at the C-terminus of proteins and holds significant value in proteomics research. Compared with traditional N-terminal sequencing, C-terminal sequencing has emerged relatively late due to its greater technical complexity. However, the rapid advancement of mass spectrometry (MS) technologies in recent years has significantly increased the importance of C-terminal sequencing based on MS in studies of protein structure and function.
Methods
MS-based C-terminal sequencing primarily relies on the high accuracy of advanced mass spectrometers, in conjunction with tailored chemical or enzymatic strategies, to enable precise C-terminal sequence analysis. The commonly employed methods include:
1. Specific Enzymatic Digestion Strategies
Because protein C-termini often lack conserved sequence motifs, employing specific proteases for selective cleavage is essential for effective C-terminal sequencing. For instance, certain carboxypeptidases can sequentially cleave amino acids from the C-terminus, allowing MS-based detection of the resulting fragments to deduce the C-terminal sequence. This strategy offers high specificity but may be challenged by complex protein structures or post-translational modifications.
2. Chemical Derivatization Methods
Another approach involves chemically modifying the C-terminus to enhance its detectability. C-terminal-specific derivatization reagents can be used to cap or label the terminal end, making it more readily identifiable during subsequent MS analysis. These methods improve detection sensitivity while minimizing ambiguities associated with secondary peptide fragmentation.
3. Tandem Mass Spectrometry (MS/MS) Techniques
Modern MS/MS approaches—such as high-resolution mass spectrometry (HRMS), electron transfer dissociation (ETD), and higher-energy collisional dissociation (HCD)—serve as powerful tools for C-terminal sequencing. These techniques generate extensive fragment ion profiles, enabling more accurate and detailed characterization of C-terminal amino acid sequences.
4. Protein-Level C-Terminal Sequencing
With the emergence of top-down proteomics, it is now possible to analyze C-terminal sequences directly at the intact protein level, bypassing enzymatic digestion. This approach circumvents the specificity constraints of proteases and is particularly advantageous for characterizing C-terminal regions that contain post-translational modifications (PTMs).
Applications
1. Protein Structure and Function Analysis
The C-terminal sequence plays a pivotal role in determining protein folding, stability, and intermolecular interactions. Accurate characterization of C-terminal sequences enables researchers to elucidate the relationship between protein structure and function, thereby advancing insights into biological systems.
2. Post-Translational Modifications (PTMs) Research
The C-terminal region is frequently a hotspot for post-translational modifications such as ubiquitination, phosphorylation, and glycosylation. C-terminal sequencing based on MS facilitates the identification and characterization of these modifications, supporting detailed investigations into their roles within cellular signaling pathways.
3. Quality Control of Antibodies and Biopharmaceuticals
During the development of monoclonal antibodies and recombinant protein therapeutics, verifying the integrity of the C-terminal sequence is essential for ensuring product efficacy and safety. C-terminal sequencing based on MS provides a robust approach for protein characterization and quality control, enabling the detection of unexpected cleavage events or modifications.
4. Study of Protein Degradation Mechanisms
Protein degradation pathways are often regulated by C-terminal signals. For instance, specific C-terminal sequences can dictate the intracellular half-life of proteins. C-terminal sequencing using MS technologies allows researchers to elucidate the molecular underpinnings of protein turnover, offering valuable perspectives for understanding disease-associated degradation mechanisms.
C-terminal sequencing based on MS has witnessed notable advancements in recent years. However, technical challenges such as the low abundance of C-terminal peptides, sequence heterogeneity, and interference from post-translational modifications persist. Future directions include the development of more efficient enzymatic digestion protocols, refinement of chemical derivatization strategies, and the integration of artificial intelligence algorithms to enhance MS data interpretation. Furthermore, innovations in single-cell proteomics and ultra-high-resolution MS platforms are expected to open new avenues for C-terminal analysis. MtoZ Biolabs offers specialized N- and C-terminal protein sequencing services, designed to address project-specific challenges and accelerate proteomics research with high analytical rigor and reliability.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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