Protein C-Terminal Sequencing: Methods, Challenges, and Optimization
Protein serves as a central executor of biological functions, with its structure intricately linked to its function. While extensive efforts have been devoted to the sequencing of protein N-termini, studies on the C-terminus (carboxyl terminus) have comparatively lagged behind. In reality, the C-terminal region plays critical roles in regulating protein stability, subcellular localization, signal transduction, and degradation. Notably, C-terminal sequencing information has demonstrated distinct value in areas such as post-translational modifications, non-canonical splicing events, and disease biomarker discovery.
How can the C-terminus of a protein be precisely characterized? What are the technical obstacles, and how might they be addressed? This review provides a comprehensive analysis of these questions.
Methods
The accurate identification and enrichment of C-terminal peptides is essential for protein C-terminal sequencing. Current mainstream approaches can be broadly classified into three categories: enzymatic digestion, chemical labeling, and direct mass spectrometry-based identification.
1. Enzymatic Digestion
This strategy involves the sequential cleavage of amino acid residues from the protein C-terminus using carboxypeptidases, followed by mass spectrometry-based monitoring of the released amino acids to infer the original sequence. The method is straightforward and well-suited for shorter peptide sequences; however, it is highly sensitive to protein conformation and post-translational modifications, which can significantly affect its efficiency.
2. Chemical Labeling and Enrichment Methods
To achieve accurate identification of C-terminal peptides in complex samples, chemical labeling techniques are frequently employed to improve detection specificity. Common approaches include:
(1) TMPP-AcOSu labeling: The chemical reagent TMPP (Trimethylammoniumbutyryl) selectively targets the C-terminus, thereby enhancing signal intensity in mass spectrometry analysis;
(2) Solid-phase enrichment: The C-terminus of peptides is covalently linked to a solid-phase medium, followed by elution of unbound fragments, allowing selective retention of C-terminal peptides;
(3) Heterobifunctional crosslinker-assisted capture: Specific crosslinkers (e.g., Biotin-NHS) are used to covalently bind C-terminal residues to affinity carriers, enabling pull-down purification of C-terminal peptides.
These strategies significantly enhance the sensitivity of C-terminal sequencing signal detection and facilitate the identification of low-abundance C-terminal peptides under complex sample conditions.
3. Mass Spectrometry-Based Direct Sequencing Methods
High-resolution mass spectrometry platforms (e.g., Orbitrap, FTICR, or TOF) enable direct MS/MS fragmentation of intact proteins or proteolytic peptides, from which C-terminal sequences can be reconstructed by analyzing the resulting fragment ions. This approach is typically integrated with database searching or de novo sequencing algorithms, making it suitable for characterizing proteins with unknown sequences or samples lacking prior annotation.
Moreover, employing specific proteases (such as LysargiNase or GluC) in a “semi-specific digestion” strategy can substantially improve C-terminal peptide coverage.
Challenges
Despite ongoing advancements, C-terminal sequencing still faces several fundamental challenges:
1. Low yield of C-terminal peptides: Conventional proteolytic digestion protocols favor the generation of N-terminal peptides, resulting in underrepresentation of C-terminal fragments;
2. Limited availability of C-terminal-specific proteases: Commonly used enzymes, such as trypsin, exhibit poor specificity for cleaving at the C-terminus;
3. Complex and heterogeneous C-terminal modifications: Post-translational modifications—including acylation, isomerization, and cyclization—can interfere with fragmentation patterns and hinder sequence interpretation;
4. High sample complexity: Abundant non-target peptides in biological samples can obscure C-terminal signals and reduce detection sensitivity;
5. Lack of specialized data analysis tools: Existing bioinformatics tools are primarily optimized for N-terminal sequencing, often resulting in suboptimal performance in C-terminal peptide identification.
Optimization Strategies
To address the aforementioned challenges, researchers have proposed several optimization strategies aimed at enhancing the accuracy and efficiency of protein C-terminal sequencing:
1. Multi-enzyme synergistic digestion: Combining proteases with distinct cleavage specificities, such as Trypsin, GluC, and AspN, to increase the relative abundance of C-terminal peptides;
2. Implementation of C-terminal enrichment strategies: Employing techniques such as solid-phase extraction, affinity capture, or isotope labeling to selectively isolate C-terminal peptides;
3. Utilization of high-resolution mass spectrometry platforms: Leveraging instruments like the Thermo Orbitrap Fusion Lumos to substantially enhance detection sensitivity;
4. Integration of AI-assisted data analysis: Applying machine learning algorithms to recognize fragment ions characteristic of C-terminal sequences, thereby improving identification accuracy;
5. Optimization of sample pretreatment workflows: Using mild processing conditions to minimize the degradation or loss of C-terminal modifications during sample preparation.
Protein C-terminal sequencing plays a vital role in elucidating protein structure and function, investigating post-translational modifications, and advancing biopharmaceutical research. With ongoing methodological advancements and technological progress, both the accuracy and the scope of applications are expected to improve significantly. MtoZ Biolabs provides accurate and rapid protein N/C-terminal sequencing services to help researchers comprehensively understand the relevant content.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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