LC-MS/MS Based C-Terminal Sequencing: Technical Advances and Challenges
One of the central objectives of proteomics is to elucidate the complete amino acid sequences of proteins. In this context, C-terminal sequencing has attracted increasing attention in recent years due to its critical role in understanding protein modifications, degradation pathways, and biological functions. The rapid advancement of liquid chromatography–tandem mass spectrometry (LC-MS/MS) has offered powerful analytical capabilities for C-terminal sequencing. However, challenges remain due to the limited availability of C-terminal-specific proteases, the complexity of fragmentation patterns, and difficulties in data analysis. This review summarizes recent technical advances in LC-MS/MS based C-terminal sequencing, and critically discusses the key challenges currently hindering its broader application.
Technical Advances in LC-MS/MS Based C-Terminal Sequencing
LC-MS/MS has become the standard method for protein sequencing, owing to its ability to combine high-resolution separation with precise mass spectrometric identification of peptides resulting from enzymatic digestion. For C-terminal sequencing, four main strategies are currently employed:
1. C-terminal Specific Enzymatic Digestion
A primary obstacle in C-terminal sequencing lies in the scarcity of efficient proteases that specifically cleave at the C-terminus. In contrast to N-terminal digestion, few enzymes—such as carboxypeptidase B and carboxypeptidase Y—are capable of sequentially trimming amino acid residues from the protein C-terminus to reveal sequence information. Nevertheless, the applicability of this approach is constrained by the substrate specificity and catalytic efficiency of these enzymes. Moreover, post-translational modifications at the C-terminus (e.g., amidation, lipidation) may interfere with enzymatic cleavage, further complicating sequencing efforts.
2. Chemical Degradation Methods
Chemical approaches offer an alternative route for C-terminal sequencing, including modified Edman-type reactions and carboxyl-activation-based strategies. Certain chemical reagents can selectively modify the C-terminal carboxyl group, facilitating stepwise identification of terminal residues. However, these methods often suffer from limited selectivity and reaction efficiency, making them unsuitable for complex protein mixtures.
3. Mass Spectrometry-Based Fragmentation Strategies
Fragmentation techniques in mass spectrometry are central to C-terminal sequencing. Commonly employed methods include collision-induced dissociation (CID), electron transfer dissociation (ETD), and higher-energy collisional dissociation (HCD). Optimizing these fragmentation modes enhances the sequence coverage of C-terminal peptides. For instance, ETD preserves labile post-translational modifications, while HCD improves the intensity of y-ion series derived from C-terminal peptides. Additionally, hybrid techniques such as electron-transfer/higher-energy collision dissociation (EThcD), which integrate the benefits of ETD and HCD, have further improved the detection sensitivity for C-terminal peptides.
4. Labeling and Enrichment Techniques
To improve the detection sensitivity of C-terminal peptides, various chemical labeling and enrichment strategies have been developed. C-terminal-specific chemical tags can selectively label the terminal carboxyl group, thereby enhancing the ionization efficiency during MS analysis. Furthermore, solid-phase enrichment approaches—such as affinity purification using streptavidin–biotin interactions—enable selective isolation of labeled C-terminal peptides, significantly improving their detection and sequencing efficiency.
Key Challenges in LC-MS/MS Based C-Terminal Sequencing
1. Sample Complexity and C-Terminal Modifications
Protein samples often contain a wide variety of proteins, and C-terminal sequences tend to be less stable, leading to the inherently low abundance of C-terminal peptides. Furthermore, post-translational modifications (PTMs) at the C-terminus—such as phosphorylation and glycosylation—can hinder enzymatic digestion and complicate MS-based detection, thereby increasing the difficulty of sequencing. Consequently, enhancing the selectivity of sample pretreatment methods and developing effective strategies for the removal or preservation of C-terminal modifications are crucial areas for future research.
2. Limitations of C-Terminal Specific Enzymes
Current enzymatic approaches for C-terminal cleavage are constrained by the limited substrate specificity and catalytic efficiency of available proteases. Thus, advancing this field requires the development of novel C-terminal-specific enzymes or the optimization of reaction conditions for existing ones. Additionally, enzyme engineering through genetic modification offers a promising route to enhance both the specificity and stability of proteases, thereby improving the overall efficiency and success rate of C-terminal sequencing.
3. Challenges in Mass Spectrometry Data Interpretation
Due to the inherently weak signals of C-terminal peptides and potential interference from co-eluting or overlapping peptides, data interpretation remains a major bottleneck in LC-MS/MS based C-terminal sequencing. Conventional database search methods often fall short in accurately identifying C-terminal sequences, underscoring the need for dedicated bioinformatics algorithms. Recent advances combining machine learning and deep learning approaches show promise in building more robust models for C-terminal peptide identification, thereby enhancing the reliability of sequencing outcomes.
4. Integration of Multiple Technologies
Given that no single approach can comprehensively address all the challenges in C-terminal sequencing, future developments will likely depend on the integration of complementary strategies. For example, the combination of chemical labeling, specific enzymatic digestion, peptide enrichment, and state-of-the-art mass spectrometry techniques can substantially improve sequencing success rates. Moreover, emerging platforms such as single-molecule sequencing and nanopore-based technologies may offer novel solutions for C-terminal sequencing in complex biological systems.
The convergence of efficient proteolysis, advanced chromatographic separation, high-resolution mass spectrometry, optimized fragmentation techniques, and AI-driven data analysis has greatly enhanced the sensitivity and precision of LC-MS/MS based C-terminal sequencing. Nevertheless, significant challenges persist, including the detection of low-abundance C-terminal peptides, prevention of non-specific degradation, handling of complex datasets, and interpretation of C-terminal PTMs. Looking forward, the integration of innovative proteases, single-cell analytical techniques, and AI-assisted bioinformatics is expected to further advance the applications of C-terminal sequencing in proteomics and precision medicine. MtoZ Biolabs utilizes advanced instrumentation and technologies to offer professional N- and C-terminal protein sequencing services.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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