Principle of N-Terminal Sequencing

The N-terminal sequence of proteins is significant in many biological processes, such as protein processing, localization, and degradation. Traditional methods like Edman degradation have been widely used but possess limitations. Modern mass spectrometry techniques offer new methods for N-terminal sequencing with higher sensitivity and resolution.

 

Sample Preparation

The first step in N-terminal sequencing is sample preparation. Protein samples are typically obtained through cell lysis and protein extraction, followed by purification. To ensure accurate sequencing, samples undergo enzymatic digestion, commonly using enzymes like trypsin and Glu-C. The resulting peptides are used for subsequent analysis.

 

Chemical Reactions

In the Edman degradation method, the N-terminal amino acid reacts with phenylisothiocyanate (PITC) to form a PTH derivative, cleaved from the peptide chain under acidic conditions. Iterative degradation cycles sequentially identify each N-terminal amino acid. However, this method has limitations with long peptides and complex mixtures.

 

Separation and Identification

Modern mass spectrometry techniques such as liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) effectively separate and identify N-terminal peptides. In LC-MS, peptides are separated by liquid chromatography and detected by mass spectrometry. MS/MS generates fragment ions through collision-induced dissociation (CID), and the mass-to-charge ratio (m/z) of these fragment ions provides peptide sequence information.

 

Data Analysis

The final step in N-terminal sequencing is mass spectrometry data analysis. Specialized software, such as Mascot or Sequest, compares experimental data with known sequences in databases. By matching the m/z of fragment ions, the peptide sequence is determined, deducing the protein's N-terminal sequence.

 

Although traditional Edman degradation still has applications, modern mass spectrometry techniques are becoming the primary method for N-terminal sequencing due to their efficiency and accuracy. MtoZ Biolabs provides integrate N-Terminal sequencing service.