Methods for C-Terminal Sequence Analysis in the Proteomic Era
With the advancement of proteomics, the analysis of C-terminal sequences has become increasingly critical. The C-terminus plays a pivotal role in determining protein function, stability, subcellular localization, and interactions, and is frequently regulated by post-translational modifications (PTMs) or specific proteolytic cleavage. However, C-terminal sequencing poses greater technical challenges compared to N-terminal sequencing, primarily due to the absence of a stepwise chemical degradation approach analogous to Edman degradation. Recent progress in mass spectrometry (MS) and the refinement of bioinformatics tools has significantly enhanced the capacity to characterize protein C-termini in the proteomic era. This review summarizes the major contemporary strategies for C-terminal sequence analysis, including enzymatic digestion, chemical labeling, MS-based techniques, and their applications in proteomic studies.
Limitations of C-Terminal Sequencing Methods
Traditionally, C-terminal sequence analysis has relied on carboxypeptidase digestion, wherein specific carboxypeptidases sequentially cleave amino acids from the C-terminus. The identity of the released residues is then used to reconstruct the terminal sequence. Despite its historical significance, this approach is limited by several factors:
1. Restricted Specificity of Carboxypeptidases
Various carboxypeptidases (e.g., Carboxypeptidase A, B, Y) exhibit substrate specificity, which restricts their applicability to a subset of protein substrates.
2. Interference from C-Terminal Modifications
The C-terminus is frequently modified by chemical groups such as amides, phosphate moieties, or glycans. These modifications can hinder enzymatic cleavage, resulting in sequencing failure.
3. Low Efficiency and Throughput
The stepwise hydrolysis process is inherently slow and typically yields only short peptide sequences (≤5 amino acids), which limits its utility for high-throughput proteomic applications.
In light of these constraints, the proteomic era demands more efficient and sensitive C-terminal sequencing approaches to enable large-scale characterization of protein C-termini.
Modern Methods for C-Terminal Sequence Analysis
1. Mass Spectrometry-Based C-Terminal Sequencing
Mass spectrometry has emerged as the principal analytical tool for C-terminal sequence determination in proteomics. Current MS-based strategies include Bottom-up MS, Top-down MS, and Middle-down MS.
(1) Bottom-up MS:
This approach involves enzymatic digestion of proteins with site-specific proteases, followed by peptide analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enzymes with C-terminal specificity (such as AspN or GluC) can generate peptides with termini that are more readily detected. However, a significant limitation is the potential loss of C-terminal fragments during proteolysis, which compromises sequence completeness.
(2) Top-down MS:
Top-down MS enables the analysis of intact proteins, eliminating the need for enzymatic digestion. Using high-energy fragmentation techniques (e.g., collision-induced dissociation [CID], higher-energy collisional dissociation [HCD], electron transfer dissociation [ETD]), this method generates informative fragment ions that allow for direct sequencing of the C-terminal region. Moreover, it facilitates the identification of post-translational modifications such as amidation and glycosylation. Nevertheless, the approach requires high-resolution instrumentation and is generally limited to samples of low complexity.
(3) Middle-down MS:
The middle-down strategy utilizes partial proteolysis to retain longer C-terminal fragments. This improves sequence coverage and mitigates the loss of terminal peptides observed in bottom-up workflows, offering a balanced solution for C-terminal analysis in complex proteomic samples.
2. Chemical Labeling Methods
Chemical labeling strategies exploit the reactivity of the C-terminal carboxyl group to selectively modify the protein C-terminus in combination with mass spectrometry analysis. In recent years, Click Chemistry has also been adopted for C-terminal labeling and applied alongside LC-MS/MS, enhancing the stability and sensitivity of detection. While these chemical labeling approaches can, under certain conditions, significantly improve the efficiency of C-terminal sequencing, their performance is often constrained by factors such as structural hindrance within proteins and incomplete reaction yields. As such, further optimization and refinement are necessary to enhance labeling efficiency and applicability.
Applications of C-Terminal Sequencing in the Proteomic Era
C-terminal sequence analysis plays a vital role in proteomics, particularly in elucidating protein degradation mechanisms, characterizing post-translational modifications (PTMs), and supporting quality control processes in biopharmaceutical development. The C-terminal sequence provides critical insights into substrate degradation patterns within the ubiquitin–proteasome system (UPS), thereby aiding in the understanding of protein stability and proteolytic pathways. For instance, in neurodegenerative disease research, C-terminal truncations of certain aberrant proteins have been closely linked to disease progression, making the characterization of their C-terminal regions essential for unraveling pathogenic mechanisms. Furthermore, post-translational modifications at the C-terminus, such as amidation or phosphorylation, serve pivotal functions in regulating protein activity. These modifications can be accurately detected using advanced mass spectrometry techniques, which greatly facilitate functional proteomics studies. In biopharmaceuticals, the integrity of the C-terminal sequence is directly associated with the biological activity and structural stability of therapeutic proteins. For example, C-terminal truncations or abnormal modifications in monoclonal antibodies (mAbs) can result in misfolded conformations, ultimately compromising therapeutic efficacy and safety. Consequently, C-terminal sequence analysis has become an indispensable tool for ensuring product consistency and maintaining quality standards in drug development pipelines.
With the ongoing advancement of proteomic technologies, C-terminal sequencing methods are expected to undergo further refinement to meet the growing demands for higher throughput and enhanced analytical sensitivity. The continued evolution of high-resolution mass spectrometry platforms, such as Orbitrap and FTICR-MS, will significantly expand the capacity to detect and characterize C-terminal peptides. Concurrently, the integration of artificial intelligence and deep learning algorithms promises to improve the accuracy of sequence identification by optimizing fragmentation prediction and peptide-spectrum matching processes. Moreover, the development of innovative chemical labeling and enrichment techniques will further boost the sensitivity of C-terminal peptide detection, facilitating their broader application in complex proteomic samples. For practical applications, services such as those offered by MtoZ Biolabs, which specialize in N- and C-terminal sequencing, provide researchers with technical solutions that can accelerate experimental workflows and enhance data reliability.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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