Application of Mass Spectrometry and Edman Degradation in Protein Sequencing

Proteins are essential functional molecules in living organisms, and studying their structure and function is crucial for understanding biological processes and disease mechanisms. Protein sequencing, the determination of the amino acid sequence of proteins, is a key step in biological research. Currently, mass spectrometry and Edman degradation are the two main methods of protein sequencing.

 

Application of Mass Spectrometry in Protein Sequencing

Mass spectrometry (MS) is a technique that analyzes and identifies molecules by measuring the mass-to-charge ratio (m/z) of ions. In protein sequencing, mass spectrometers are often combined with liquid chromatography (LC) to form liquid chromatography-mass spectrometry (LC-MS).

 

1. Principle of Mass Spectrometry Sequencing

Mass spectrometry sequencing involves ionizing the protein sample to create charged ions, which are then separated in an electric field. The detector measures the m/z values of these ions, from which the amino acid sequence of the protein can be inferred.

 

2. Application Scope

(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.

 

3. Advantages and Disadvantages

(1) Advantages

High sensitivity, high throughput, ability to analyze complex mixtures, and detection of PTMs.

 

(2) Disadvantages

Requires expensive equipment and specialized operation, and the sample preparation process is complex.

 

Application of Edman Degradation in Protein Sequencing

Edman degradation is a classical protein sequencing method that sequentially removes and identifies amino acids from the N-terminus through chemical reactions.

 

1. Principle of Edman Degradation

Edman degradation uses phenylisothiocyanate (PITC) to react with the N-terminal amino acid of the protein, forming a phenylthiocarbamoyl derivative. This compound cyclizes and cleaves from the peptide chain under acidic conditions. By repeatedly performing this reaction, the amino acid sequence of the protein can be determined step by step.

 

2. Application Scope

(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.

 

3. Advantages and Disadvantages

(1) Advantages

High precision, direct determination of the amino acid sequence, suitable for analyzing N-terminal modifications.

 

(2) Disadvantages

High purity of samples required, can only sequence from the N-terminus, cannot handle internal amino acid sequences and large proteins.

 

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. MtoZ Biolabs provides integrate protein sequencing service.