Edman Degradation vs Sanger Sequencing

Edman degradation and Sanger sequencing are both classical methods used to analyze biomolecule sequences. Although their principles and application fields are different, both of them have important biological research significance. Below I will compare the principle, application, advantages and disadvantages:

1. Comparison of principles

Edman degradation

• Objective: It is mainly used to determine the amino acid sequence of protein or peptide chain.
• Principle: Eideman degradation method gradually removes amino acids from the N-terminal of protein or peptide, cutting one amino acid at a time, and analyzing its identity by chromatography. Specifically, firstly, the N-terminal amino acid was labeled with phenylisothiocyanic acid (PITC), then the amino acid was excised under acidic conditions, and it was transformed into a stable benzothiazolyl amino acid (PTH). Finally, the types of amino acids were determined by high performance liquid chromatography (HPLC) separation and analysis.

N‐terminal Edman degradation (Zhang H et al., 2020)

Sanger sequencing

• Objective: It is mainly used to determine the nucleotide sequence of DNA or RNA.
• Principle: Sanger sequencing method interrupts the extension of DNA chain by synthesizing the DNA chain and using a special labeled nucleotide (DDNTPS) to stop the synthesis. A series of DNA fragments with different lengths were obtained by setting reaction mixtures containing different ddNTPs. Finally, these fragments were separated by capillary electrophoresis to read the DNA sequence.

C terminal and N terminal sequencing

Protein N-terminal sequencing refers to the analysis of the amino terminal (i.e. the initial part of

2. Comparison of application fields

Core application scenarios of Edman degradation method

• Short peptide sequencing: for example, The biological mineral aragonite structure of Noah's Ark L. was studied, and the mineral structure and morphological characteristics of Noah's Ark L. were characterized by X-ray diffraction, field emission scanning electron microscope (FE-SEM) and atomic force microscope (AFM). Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were used to study its thermal properties. Edman degradation analysis was used to study protein in soluble organic matrix (SOM). The results show that the biological mineral structure of Noah's Ark shell is complex, with nano-particles and micron-sized aragonite crystals with abnormal development, showing orthogonal symmetry. A small amount of organic matter (SOM) is connected with these mineral structures, accounting for 1.5% of the total mass of the shell. Through Edman degradation analysis, it was found that the shell contained tetrapeptide repetitive sequence (TPR) proteins, and these protein proteins were similar to TPR proteins in bacteria, which might play a key role in biomineralization (Sondi I et al., 2025).
• Characterization of antibody drugs: verify the N-terminal sequence consistency of monoclonal antibody complementarity determining region (CDR). MMP-14 is a key enzyme in tumor growth and metastasis and an important target of cancer treatment. Fab 3A2 antibody is easily cleaved by MMP-14 in acidic environment, which leads to its loss of inhibitory effect. In order to improve the proteolytic stability of Fab 3A2, the cleavage site was analyzed. It was found that after being incubated with MMP-14 for 12 hours at 37°C and pH 6.0, Fab 3A2 was significantly cleaved, resulting in about 60% of Fab 3A2 being cleaved into two fragments with different molecular weights-13 kDa and 36 kDa. By studying the cleavage fragment, it is speculated that cleavage occurred between the heavy chain and the light chain of Fab, resulting in LC-C H1 fragment and VH fragment with truncated N-terminal. Further analysis showed that the 36 kDa fragment represented the LC-C H1 fragment, while the 13 kDa fragment was the truncated region of the heavy chain. The N-terminal amino acid sequence of the 14 kDa fragment was determined by Edman degradation, and it was found to match the specific sequence of Fab 3A2 heavy chain (located in the CDR-H3 region), which confirmed that the cleavage did occur between the residues in the CDR-H3 region. That is, it was found that MMP-14 was cleaved between N100h(P1) and L100i(P1') residues in the CDR-H3 region of Fab 3A2, resulting in the loss of inhibitory function of the antibody. The mutation of P1'(L100i) causes the antibody to lose its inhibitory effect, while the mutation at P1(N100h) or P3'(A100k) effectively improves the stability and inhibitory activity of the antibody (Lee KB et al., 2019).
• Posttranslational modification (PTM) localization: detection of N-terminal acetylation. The organelle sprayed by green microalgae Pyramimonas parkeae contains a banded structure, which can be released from the cell when it is stimulated and form a tubular structure. Through Tricine-SDS-PAGE, Edman degradation and LC/MS/MS analysis, it was found that this banded structure contained some protein and low molecular acid polymers. Protein in the banded structure was identified as the core histone (H3, H2A, H2B and H4), and the polysaccharide part was mainly composed of β(1-4) linked polymers containing N- acetylglucosamine. That is, the banded structure of P. parkeae is mainly composed of core histone and polymer complex containing N- acetylglucosamine (Yamagishi T et al., 2015).
• Auxiliary analysis of disulfide bonds: tracking cysteine pairing. The blood cell extract of red king crab (Paralithodes camtschaticus) was extracted to explore its potential as a new source of antimicrobial peptides. Three peptides rich in cationic cysteine (Cys) were successfully isolated by bioassay, and they were named Osteocin 1-3. Edman degradation and high-resolution tandem mass spectrometry showed that these peptides were between 38 and 51 amino acids in length, and they shared a unique Cys motif consisting of eight cysteines, forming four disulfide bonds. The arrangement patterns of these disulfide bonds (Cys1-Cys8, Cys2-Cys6, Cys3-Cys5 and Cys4-Cys7) have not been reported in previous antibacterial peptides, and they may belong to a new family of antibacterial peptides rich in Cys (Moe MK et al., 2018).

Core application scenarios of Sanger sequencing method

• Diagnosis of genetic diseases: The genetic characteristics of cystic fibrosis (CF) in China patients are different from those in Caucasian patients. By sequencing all exons, the known variation of CFTR gene c.1000C > T, p.R334W was found in the proband of family I, which has been reported as a pathogenic variation in the literature. A new CFTR mutation c.1409T > A, p.V470E was found among the probands IIa and IIb of family II, which has not been reported in the literature or appeared in the cystic fibrosis mutation database. Through the prediction of SIFT and CADD tools, this new mutation is considered to be possibly pathogenic. Furthermore, the variation found in the whole exon sequencing was verified by Sanger sequencing, and the variation of each family was separated and analyzed. Sanger sequencing confirmed the variation in proband, and healthy parents all carried heterozygous variation at corresponding positions (Yang B et al., 2021).
• Tumor-driven mutation screening: The mutation of EGFR in circulating tumor DNA(ctDNA) in plasma of patients with lung cancer was detected by using the Sanger method of BDA. The study included 195 plasma samples from patients with lung cancer. The BDA Sanger method can detect the EGFR mutation of 0.20% VAF (variant allele frequency) in plasma samples. In newly treated patients, the consistency between BDA Sanger method and tissue and plasma samples was 79%. Super-ARMS detected EGFR mutation in 34.4% of the samples, while BDA Sanger detection was detected in 41.0% of the samples. The overall consistency of the two methods in detecting EGFR mutation is 82%. The BDA Sanger method can also detect some rare EGFR mutations, which Super-ARMS did not find (Jiang H et al., 2022).
• Synthetic biology: plasmid construction verification (such as gRNA sequence confirmation of CRISPR vector). CRISP-ID is a newly developed method for directly identifying insertional deletions (Indels) from Sanger sequencing data, especially in multiplex PCR products, which solves the problem of overlapping spectra. In order to verify the validity of CRISP-ID, researchers used CRISPR-Cas9 technology to knock out some genes: ELOVL6, MBTPS1 and SREBF1 genes in diploid human cell lines, Elovl6 genes in diploid mouse derived cell lines and Fxr1 genes in mouse in vivo models. In Sanger sequencing data, occasional small errors (such as poor peak quality or random insertion/replacement) have no effect on the determination of the size and position of insertion deletion. CRISP-ID can successfully and efficiently identify insertion deletion, and the result is completely consistent with the traditional single colony method (Dehairs J et al.,2016).
• Microbial identification: 16S rRNA gene sequencing (bacterial species classification). 16S rRNA gene sequencing is widely used to identify infections caused by bacteria, especially when conventional phenotypic identification methods fail. It is particularly effective in identifying uncommon or rare bacteria and can reliably determine the genus and species level of bacteria. 16S/ITS rRNA gene polymerase chain reaction (PCR) and Sanger sequencing were used to identify the pathogen of mycotoma, and 16S/ITS rRNA sequencing was directly performed on grain samples to identify the pathogen. Among 15 black particle samples, 13 samples (86%) were successfully sequenced, and 11 samples (73%) identified the fungal pathogen Madurella mycetomatis, which was caused by mycotoma. Cladosporium sphaerospermum was found in a sample, which is a possible new pathogen. Among the 12 red particle samples, 6 samples (58.3%) were successfully sequenced, and Actinomadura pelletieri was found. These samples also helped to identify two possible actinomycetes pathogens, A. madurae and A. geliboluensis. In general, the pathogen of mycetoma was successfully detected and identified in 59.4% of cases by 16S/ITS rRNA sequencing technology (Diongue K et al., 2024).
• Forensic medicine: short tandem repeat (STR) typing. STR is an important tool in personal identification and paternity testing, but genotyping errors may have a negative impact on forensic identification. In this study, it was found that there was an invalid allele at D2S1338 locus in paternity test, which led to the failure to conform to the genetic laws between father and daughter, and thus the paternity may be wrongly excluded. In order to find out the reason, a new primer was used for re-amplification, and a large fragment of it was sequenced by Sanger. Through this analysis, we found a change that may lead to problems: a change from G to G/T occurred in the 59 base positions at the 3' end of the core repeat sequence of D2S1338. This change may be a new variant of primer binding region, which has not been reported yet. It was also found that there were multiple single nucleotide polymorphisms (SNPs) in the upstream and downstream sequences of D2S1338, and the minor allele frequency (MAF) of these SNPs was greater than 0.1. These SNPs may affect the amplification reaction and lead to wrong genotyping results (Jiang L et al., 2023).

3. Technical advantages and limitations

Advantages and limitations of edman degradation method

• Superiority: Single amino acid level accuracy, can identify isomers (such as isoleucine and leucine). Direct identification of N-terminal modifications (such as methionine oxidation). A single operation can complete 30-50 cycles (about 1-2 days).
• Limit: C-terminal or internal sequence cannot be obtained directly, and only N-terminal sequence can be parsed. It is completely ineffective for N-terminal blocking proteins (such as acetylation). Side chain modifications such as glycosylation and phosphorylation cannot be analyzed. Long-chain proteins need to be cut by graded enzymes, which takes several weeks.

Advantages and limitations of Sanger sequencing method

• Superiority: Single base resolution, gold standard verification technology (error rate < 0.001%). It can detect heterozygous variation as low as 5% mutation frequency (such as tumor samples). Compatible with genomic DNA, cDNA, plasmid and other samples.
• Limit: Long fragments (> 1 kb) need to be sequenced in segments, which doubles the cost. It is impossible to accurately quantify the allele ratio. Signal attenuation is easy to occur in areas with high GC content (> 70%)

4. Data output and analysis

Data flow of Edman degradation method

• Original data: RP-HPLC chromatogram (PTH- amino acid retention time and peak area);
• Analysis steps: constructing PTH- amino acid standard library (containing 37 common amino acids and modified forms); Calculate the cycle efficiency of each round by peak area integration (normal value > 98%); Mass spectrometry verification (LC-MS/MS) for abnormal peaks (such as overlapping peaks);
• Software support: Procise™ software (ABI): automatic loop control and sequence splicing;
• DeNovo®: Combined with mass spectrometry data to assist the analysis of complex modifications.

Data flow of Sanger sequencing method

• Raw data: fluorescence signal trace of capillary electrophoresis (.ab1 file);
• Analysis steps: using Phred algorithm to identify bases (Q value ≥30 is high quality); Splicing overlapping fragments by Phrap/Consed software; Compare with reference sequence (such as BLAST) to detect mutation;
• Automation tools: SeqScanner(Thermo Fisher): automatic generation of consensus sequence; Mutation survey: visual detection of insertion/deletion and SNP.

5. Technological development and substitution

Alternative technology and complementary strategy of Edman degradation

• Mass spectrometry technology: Shotgun: LC-MS/MS analysis after enzyme digestion, suitable for long-chain proteins (such as antibody full-length sequences); Top-down MS: the complete protein is directly analyzed, but the resolution is limited (> 30 kDa is difficult);
• Preserve the scene: N-terminal exploration of unknown protein of new species (without database); Identification of truncated synthetic peptides (such as GLP-1 analogues);

Alternative technology and collaborative application of Sanger sequencing

• Next generation sequencing (NGS): Illumina short reading length: whole genome sequencing (WGS), exon sequencing (WES); PacBio SMRT: long reading length (> 10 kb) to solve structural variation;

• Preserve the scene: NGS result verification (such as Sanger confirmation necessary for clinical diagnosis); Low complexity samples (such as plasmids and PCR products);

References

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2. Lee KB, Dunn Z, Ge X. "Reducing proteolytic liability of a MMP-14 inhibitory antibody by site-saturation mutagenesis." Protein Sci. 2019 Mar;28(3):643-653. doi: 10.1002/pro.3567
3. Yamagishi T, Kurihara A, Kawai H. "A Ribbon-like Structure in the Ejective Organelle of the Green Microalga Pyramimonas parkeae (Prasinophyceae) Consists of Core Histones and Polymers Containing N-acetyl-glucosamine." Protist. 2015;166(5):522-33. doi: 10.1016/j.protis.2015.08.003
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7. Dehairs J, Talebi A, Cherifi Y, Swinnen JV. "CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing." Sci Rep. 2016;6:28973. doi: 10.1038/srep28973
8. Diongue K, Dione JN, Diop A, Kabtani J, Diallo MA, L'Ollivier C, Seck MC, Ndiaye M, Badiane AS, Ndiaye D, Ranque S. "Direct 16S/ITS rRNA Gene PCR Followed by Sanger Sequencing for Detection of Mycetoma Causative Agents in Dakar, Senegal: A Pilot Study Among Patients with Mycetoma Attending Aristide Le Dantec University Hospital." Mycopathologia. 2024 ;189(5):80. doi: 10.1007/s11046-024-00891-w
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