C-Terminal vs N-Terminal Sequencing: Key Differences, Methods, and Applications

Protein terminal sequencing encompasses complementary approaches for analyzing amino acid sequences at both polypeptide termini. For C-terminal characterization, enzymatic digestion via carboxypeptidases or chemical cleavage coupled with mass spectrometry (MS) enables progressive residue removal from the carboxyl terminus. These methodologies facilitate the determination of terminal sequences, identification of translational termination sites, and detection of PTMs, including ubiquitination and phosphorylation. The approaches are particularly valuable for investigating protein interaction interfaces and structural stability while accommodating modifications like C-terminal amidation or truncation mutations.

N-terminal sequencing predominantly employs Edman degradation or MS-based peptide enrichment strategies. These techniques elucidate initiation sequences critical for understanding protein folding, functional domains, and PTM patterns such as acetylation or formylation. The acquired N-terminal data further assist in mapping transcriptional initiation points and gene structure annotation.

This article describes the sequencing techniques of both and the various differences.

Core difference

C-Terminal Analysis

• Methodology: Carboxypeptidase digestion or chemical cleavage-MS integration
• Analytical Focus: Molecular interaction surfaces, stability determinants, and PTM localization
• Detection Capacity: Amidation, truncation variants, and splice isoforms
• Specimen Requirements: Intact proteins (enzymatic approach) or proteolytic fragments (MS-based)
• Resolution: 3-5 residue resolution (enzymatic) or sequence-dependent coverage (MS)

N-Terminal Analysis

• Methodology: Cyclic Edman degradation or N-terminal peptide enrichment-MS
• Analytical Focus: Biosynthetic processing, degradation signals, and PTM regulation
• Detection Capacity: Acetylation, formylation, and ubiquitination patterns
• Specimen Requirements: Highly purified proteins (Edman) or digested peptides (MS)
• Resolution: 15-30 residue determination (Edman) or full-sequence profiling (MS)

Protein C-terminal sequencing

Protein C-terminal sequencing aims to analyze the aa sequence of carboxyl terminal of protein, and its technical strategies are mainly divided into three categories: carboxypeptidase method, chemical cleavage method and tandem MS.

Carboxypeptidase method

Free amino acids are released from C-terminal by using the enzymatic activity of carboxypeptidase (such as carboxypeptidase Y and carboxypeptidase P), and the sequence is deduced by monitoring the release sequence.

• Carboxypeptidase A(CPA): Substrate specificity: C-terminal aromatic (Phe, Tyr) and neutral aliphatic (Ala, Val) amino acids are preferentially cleaved; Metal dependence: Zn²+ is needed as a cofactor (the activity is inhibited by EDTA);
• Carboxypeptidase B(CPB): Substrate specificity: specific cleavage of basic amino acids (Lys, Arg); Clinical application: C-terminal Lys residue detection of recombinant protein drugs (such as C-terminal homogeneity verification of antibody drugs).
• Carboxypeptidase Y(CPY, fungal origin): Broad spectrum activity: it can cut all amino acids except Pro; Advantages: 6 M urea tolerance, suitable for some denatured proteins.
• Sample pretreatment: protein is denatured (such as urea treatment) to expose the C-terminal.
• Enzymatic hydrolysis reaction: under the optimum pH and temperature (such as carboxypeptidase Y: pH 5.5, 37℃), samples were taken in different periods.
• Detection of amino acids: Use high performance liquid chromatography (HPLC) or amino acid analyzer to quantify the released amino acids, and deduce the C-terminal sequence according to the time gradient (usually 3-5 residues can be resolved).
• Limitation: the efficiency of enzyme digestion is affected by amino acid types (for example, Pro and Arg may block enzyme digestion), and milligram samples are needed.
• Advantages: it is suitable for whole protein without prior enzyme digestion; 3-5 residues can be resolved.
• Spatial steric hindrance problem: Pretreatment: 2% SDS was used to partially denature the protein and expose the C-terminal;
• Post-translational modification interference: Glycosylation: first deglycosylation with PNGase F; Phosphorylation: alkaline phosphatase treatment;

C-terminal peptide enrichment workflow through the PBC strategy (Zhai L et al., 2022)

Chemical cracking method

C-terminal peptide bonds were specifically cleaved by chemical reagents, and the remaining peptide fragments were analyzed by MS.

• Akabori reaction: protein was hydrolyzed by hydrazine to form C-terminal hydrazide derivatives, and the truncated peptide was detected by MS to determine the C-terminal sequence.
• Trimethylsilyl diazomethane (TMSD) labeling: C-terminal carboxyl group was derivatized to enhance the mass spectrum signal.
• Steps: After hydrazine hydrolysis or derivatization, the molecular weight difference of products was analyzed by MS (such as MALDI-TOF).
• Classical chemical cracking strategy: Bromide cyanide (CNBR): Me T-X peptide bond (X≠Pro). Hydroxylamine (NHOH): ASN-Gly peptide bond. Bpns-skatole: Trp-x peptide bond.
• Emerging chemical tools: Thio-anhydride method (TA-Cl): Selectively modify C-terminal carboxyl group, and gradually release amino acids through Edman-like cycle; Advantages: no need for enzyme digestion, compatible with N-terminal blocking protein; Photo-activated probe (such as APEX2): C-terminal peptide was enriched by proximity labeling technique for mass spectrometry analysis.
• Risk control: Side effect management:CNBr may oxidize Cys: adding 10 mM DTT to protect sulfhydryl; Deamidation caused by hydroxylamine: control the reaction time less than 4 hours.
• Advantages: it can handle modified C-terminal (such as amidation); Compatible with low purity samples.
• Limitations: There are many side reactions, and the reaction conditions need to be strictly optimized.

Tandem mass spectrometry

Protein was hydrolyzed into a peptide mixture, and the C-terminal peptide ions were selectively captured by tandem MS (MS/MS), and the fragment ion spectrum was analyzed to determine the sequence.

• Enzymatic digestion strategy: use protease such as Lys-C or Asp-N to generate C-terminal peptide suitable for MS analysis.
• C-terminal peptide enrichment: selective separation of C-terminal peptide based on charge or chemical labeling (such as strong cation exchange chromatography).
• Mass spectrometry: Screening parent ions by primary MS; Secondary MS (such as electron transfer dissociation ETD) preferentially breaks the C-terminal peptide bond to generate Y-ion series; Combining with database search tools (such as Mascot and MaxQuant) to match theoretical sequences.
• C-terminal specific enrichment strategy: Chemical marking method: C-TAILS (C-Terminal Amine Isotope Labeling)：N-terminal amino group of protein was labeled with O- methylisourea, and only C-terminal peptide contained free α-amino group after pancreatin digestion, which was enriched by strong cation exchange (SCX) column. Enzymatic auxiliary method:Carboxypeptidase y digestion: removing non-C-terminal peptide segments and retaining intact C-terminal peptide.
• Data analysis algorithm breakthrough: C-terminal specific database search: Software: MaxQuant's C-TERMINI module, FragPipe-CTerm; Parameter: set the restriction rule as "no C-terminal restriction"; Deep learning assistance: Tool: DeepCterm (predicting C-terminal peptide based on convolutional neural network); Accuracy: the identification sensitivity of modified peptide was improved to 90%.
• Advantages: PTMs (such as amidation and phosphorylation) can be analyzed and the sensitivity reaches the femtomolar level.

Protein N-Terminal Sequencing

N-terminal sequencing technologies enable precise determination of amino-terminal polypeptide sequences through three principal methodologies: Edman degradation, reverse transcription PCR (RT-PCR), and mass spectrometry (MS).

Edman Degradation Approach

This technique employs phenylisothiocyanate (PITC) to sequentially derivatize and cleave N-terminal residues.

Workflow

• Sample Preparation: Purified proteins are resolved via SDS-PAGE and electroblotted onto PVDF membranes. Target bands are excised following Coomassie staining.
• Cyclic Reactions:
  • Coupling: PITC reacts with α-amino groups to form phenylthiocarbamoyl derivatives.
  • Cleavage: Trifluoroacetic acid liberates the terminal residue as a thiazolinone derivative.
  • Conversion: Acidic hydrolysis stabilizes residues into phenylthiohydantoin (PTH)-amino acids.
  • Identification: HPLC separates PTH derivatives, with retention times matched to standards.
• Iteration: Sequential cycles typically resolve 15–30 residues.
• Strengths: High accuracy for purified proteins.
• Limitations: Incompatible with N-terminal blocked proteins or complex mixtures.

RT-PCR-Based Sequencing

This indirect method infers N-terminal sequences from mRNA-derived cDNA.

• RNA isolation and reverse transcription generate cDNA.
• PCR amplifies the 5' coding region of target genes.
• Sanger sequencing translates DNA data into amino acid sequences.
• Constraints: Fails to detect post-translational modifications (PTMs) or somatic mutations.

Mass Spectrometric Strategies

Two complementary MS workflows enable direct N-terminal profiling:

Top-Down MS

• Intact proteins are ionized (e.g., electrospray ionization) and fragmented via electron-based dissociation (ECD/ETD), preserving N-terminal ions for de novo sequencing.

Bottom-Up MS

• Enzymatic digestion (e.g., trypsin) generates peptides.
• Chemical blocking of internal lysine ε-amines enriches N-terminal peptides.
• LC-MS/MS analysis with N-terminal-specific algorithms identifies sequences and PTMs (e.g., acetylation, pyroglutamylation).
• Advantages: Detects PTMs, achieves nanogram sensitivity, and handles complex samples.

Technically different

• Mainstream technologies: C-terminal sequencing is tandem MS and carboxypeptidase; N-terminal sequencing is MS and Edman degradation.
• Resolution: 3-20 amino acids (higher by MS); 15-30 amino acids (Edman method) or full length (MS).
• Sample requirements: milligrams (enzyme/chemical method), nanograms (MS); micrograms (Edman method), nanograms (MS).
• Modification compatibility: detects amidation, glycosylation; detects acetylation, formylation, etc.

Different goals

• C-terminal sequencing: C-terminal sequencing can resolve protein folding or stabilize three-dimensional structures involving C-terminal residues (e.g. hydrophobic residues); reveal protein-protein and protein-nucleic acid interactions involving C-terminal binding domains (e.g. ligand binding at the C-terminal end of the SH3 structural domain); and modifications such as C-terminal amidation and glycosylation (e.g. hormone activity is dependent on C-terminal amidation).
• N-terminal sequencing: N-terminal sequencing resolves N-terminal sequences containing signal peptides (guiding subcellular localization) and their cleavage sites (determining mature protein forms); reveals N-terminal modifications such as acetylation and formylation affecting protein stability, interactions, and degradation (e.g., N-terminal rule pathway); and elucidates part of the N-terminal enzyme activity centers or binding sites (e.g., catalytic kinase structural domains).

Different application backgrounds

C-terminal sequencing

It is suitable for studying the C-terminal specific modification, interaction and function of protein.

It is often used for C-terminal directional modification or variation analysis of functional protein.

• C-terminal sequencing (analysis of protein's C-terminal) is very important in protein's functional annotation and proteolysis research. However, due to the low reactivity of C-terminal, the research progress of C-terminal omics is slow, far behind N-terminal omics. The researchers developed a negative selection strategy-CPB-Chafradic, which cleaves protein by carboxypeptidase B (a highly reactive enzyme) and reduces the charge state of the non-C-terminal peptide, so that the C-terminal peptide can be separated and analyzed more easily.Combined with efficient fractional diagonal chromatography based on charge, C-terminal peptide can be effectively separated, thus enhancing the detection ability of C-terminal. Using this strategy, the researchers identified 441 classical C-termini and 510 new C-termini in E.coli cell lysate. These new findings represent 2 times and 5.8 times of the traditional methods, respectively. Through the parallel digestion of trypsin and LysC, the researchers further identified 604 classical C-terminal and 818 new C-terminal, and these new findings greatly expanded the C-terminal data set of E.coli.. In Jurkat cells, 107 cleavage sites of caspase-3 and 102 substrates were identified by this strategy, which indicated that this strategy has important application potential in studying protein hydrolysis, especially Caspase-3 cleavage (Chen L et al., 2020).
• Proteases are the second largest enzyme group in human body, which play an important role in many physiological processes and are considered as potential targets for the treatment of malignant tumors. In this study, C-TAILS was proposed. The core of this technology is to label and identify the C-terminal peptide, that is, the peptide segment produced after the end of protein molecule was cut. The application of C-TAILS method in metabolically labeled cancer cell lines can identify C-terminal peptide more effectively and provide an important tool for cancer-related protein omics research (Solis N et al., 2018).
• The C-terminal region of proteins is crucial for numerous biological functions, necessitating precise analytical methods to elucidate its structural and functional roles. To address this, the C-TAILS (C-Terminal Amine-based Isotope Labeling of Substrates) approach was implemented, which specifically targets protease-cleaved C-terminal peptides. In this study, amidation reaction conditions were systematically optimized to maximize the generation of fully amidated derivatives, thereby improving peptide labeling efficiency and detection sensitivity.A key modification involved replacing dimethylation with acetylation for lysine residue blocking, which reduced non-specific interactions. This optimized protocol identified 232 C-terminal peptides in E. coli lysates, representing a 42% increase in coverage compared to standard C-TAILS workflows. Replicate experiments (n=3) using 80 μg of sample further validated the method's robustness, yielding 481 C-terminal peptides corresponding to 369 unique termini (Zhang Y et al., 2015).
• A novel computational tool termed Lys-Sequencer was engineered for de novo protein peptide sequencing. To validate its performance, bovine serum albumin (BSA) underwent complementary enzymatic digestion using Lys-C and Lys-N proteases. The resultant peptides were fractionated via reversed-phaseUPLC)and subjected to high-resolution Orbitrap mass spectrometry with CID fragmentation.The algorithm operates by pairing parent ion mass spectra and categorizing fragment ion types based on mass-to-charge (m/z) relationships, enabling sequence reconstruction and confidence scoring for each peptide. Validation studies demonstrated the identification of 34 dominant BSA peptides, resolving 391 amino acid residues with 67% sequence coverage of the full-length protein. Ambiguities affected merely 6% of residues, though amino acid composition assignments remained accurate. Notably, the algorithm achieved unambiguous sequencing of peptides up to 17 residues in length, with exceptions observed for an 18-mer containing lysine residues (Mao Y et al., 2020).
• A key problem in the study of Alzheimer's disease (AD) is the reproducibility of aggregation behavior of Aβ peptide in vitro. In order to improve the solution behavior of Aβ peptide, the solubility of Aβ peptide was improved by inserting methionine sulfoxide, or the solubility and stability were improved by adding cationic tail at the C-terminal of peptide chain. By introducing lysine residues in peptide synthesis and removing these lysine residues by immobilized carboxypeptidase B in the purification process after synthesis, higher quality and purity of Aβ42 and Aβ46 peptides were obtained.The product after chromatographic column treatment was analyzed by HPLC to confirm whether Lys residue was effectively removed and no new impurities or pollution peaks were generated. Adding reversible Lys residues and removing them with carboxypeptidase B may be a generally effective method to prepare other hydrophobic peptides, which is suitable for those sequences that do not end with arginine (Arg) or lysine (Lys). In addition, the study also pointed out that Aβ46 has stronger amyloid production ability than Aβ42 or Aβ40, and may play a role in the initial stage of the formation of A amyloid β-protein (Chemuru S et al., 2014).
• Casapse activation and protein hydrolysis and cleavage are very important early events in apoptosis. Identifying these protein substrates cleaved by caspase is helpful to understand the process of apoptosis and provide potential targets for cancer treatment. Although protein omics methods based on N-terminal marker have been used to identify protein of caspase cleavage, these methods have some limitations. ProC-TEL, as a new protein omics method, can help to identify caspase cleavage events during apoptosis. By using ProC-TEL, researchers identified many known and newly discovered caspase cleavage sites, and confirmed by biochemical means that the expression levels of various protein decreased after kinase inhibition. Through sequence analysis, ProC-TEL can identify some caspase cleavage events that traditional N-terminal methods can't (Duan W et al., 2016).

N-terminal sequencing

It is suitable for studying the synthesis, degradation, folding and other processes of protein, especially the influence on the stability and activity of protein.

In the quantitative and qualitative analysis of protein MS, N-terminal is often used as an identifier or indicator sequence to trace the source or biological pathway of protein.

For more applications of protein N-terminal sequencing, please refer to "Overview of Edman Sequencing".

Key Differences Between C-Terminal and N-Terminal Sequencing

References

1. Chen L, Shan Y, Yang C, Sui Z, Zhang X, Zhang L, Zhang Y. "Carboxypeptidase B-Assisted Charge-Based Fractional Diagonal Chromatography for Deep Screening of C-Terminome." Anal Chem. 2020;92(12):8005-8009. doi: 10.1021/acs.analchem.0c00762
2. Solis N, Overall CM. "Identification of Protease Cleavage Sites and Substrates in Cancer by Carboxy-TAILS (C-TAILS)." Methods Mol Biol. 2018;1731:15-28. doi: 10.1007/978-1-4939-7595-2_2
3. Zhang Y, He Q, Ye J, Li Y, Huang L, Li Q, Huang J, Lu J, Zhang X. "Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome." Anal Chem. 2015 ;87(20):10354-61. doi: 10.1021/acs.analchem.5b02451
4. Mao Y, Daly TJ, Li N. "Lys-Sequencer: An algorithm for de novo sequencing of peptides by paired single residue transposed Lys-C and Lys-N digestion coupled with high-resolution mass spectrometry." Rapid Commun Mass Spectrom. 2020 Feb 15;34(3):e8574. doi: 10.1002/rcm.8574
5. Chemuru S, Kodali R, Wetzel R. "Improved chemical synthesis of hydrophobic Aβ peptides using addition of C-terminal lysines later removed by carboxypeptidase B." Biopolymers. 2014 Mar;102(2):206-21. doi: 10.1002/bip.22470
6. Duan W, Chen S, Zhang Y, Li D, Wang R, Chen S, Li J, Qiu X, Xu G. "Protein C-terminal enzymatic labeling identifies novel caspase cleavages during the apoptosis of multiple myeloma cells induced by kinase inhibition." Proteomics. 2016 Jan;16(1):60-9. doi: 10.1002/pmic.201500356