Edman sequencing method analyzes the protein N-terminal sequence in sequential Edman reactions. While mass spectrometry technology has been commonly used for protein analysis, Edman sequencing is still a powerful and irreplaceable method for protein N-terminal sequencing. As a well-established sequencing method, Edman sequencing provides more accurate protein sequence data, compared to MS. 

Advantages of Edman Degradation

1. High Accuracy

One of the notable advantages of Edman Degradation is its high accuracy. By sequentially removing and identifying each amino acid, the method ensures precise sequence identification, which is crucial for determining the primary structure of proteins.

2. Low Sample Requirement

Compared to some other protein sequencing methods, Edman Degradation requires relatively small amounts of samples, making it particularly suitable for analyzing trace amounts of proteins.

3. High Degree of Automation

Modern Edman degradation instruments are highly automated, capable of processing multiple samples continuously, reducing human error and labor intensity, and increasing efficiency.

Disadvantages of Edman Degradation

1. Sample Limitations

Edman Degradation is less suitable for longer sequences. Typically, when the polypeptide chain exceeds 50 amino acids, the reaction efficiency significantly decreases, leading to loss of sequence information.

2. N-Terminal Blockage

If the N-terminal of the polypeptide chain is chemically modified or blocked, Edman Degradation cannot effectively identify the amino acid at that position. This limits the method's application on certain proteins.

3. Complex Sample Processing

For complex samples, especially mixtures containing multiple proteins, the preprocessing steps for Edman Degradation are complex, requiring protein separation and purification, which increases the time and difficulty of the experiment.

4. Sequence Limitations

The method can only sequentially degrade from the N-terminal and cannot directly obtain amino acid sequence information from the middle or C-terminal. Therefore, for research requiring comprehensive sequence information, other sequencing methods may need to be combined.

Application of Mass Spectrometry in Protein Sequencing

1. Protein Identification

MS can identify unknown proteins by matching the measured mass spectra with known protein sequences in databases.

2. Protein Quantification

Using labeling techniques (e.g., isotope labeling) and MS, proteins can be quantified absolutely or relatively.

3. Post-Translational Modifications (PTMs) Analysis

MS can detect and identify PTMs of proteins, such as phosphorylation, acetylation, and glycosylation.

Application of Edman Degradation in Protein Sequencing

1. Sequencing of Purified Proteins

Suitable for determining the N-terminal sequence of purified proteins or peptides.

2. Analysis of N-Terminal Modifications

Can analyze chemical modifications at the N-terminus, such as acetylation.

3. Confirmation of Mass Spectrometry Results

After MS analysis, Edman degradation can be used to confirm the amino acid sequence inferred by MS.

Mass spectrometry and Edman degradation are indispensable techniques in protein sequencing, each with its own strengths and weaknesses. Mass spectrometry offers high sensitivity and throughput, suitable for studying complex protein samples and post-translational modifications. Edman degradation, with its high precision and direct sequencing capability, is mainly used for determining the N-terminal sequence of purified proteins. In practice, these methods are often combined, leveraging their respective strengths to accurately resolve protein structures, thus providing crucial foundational data for biological research.