Analysis of C-Terminal Sequencing Using LC-MS/MS

C-terminal sequencing plays a crucial role in proteomics research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for identifying C-terminal sequences.

 

The C-terminal sequence of proteins is critical in many biological processes, including protein-protein interactions, cellular localization, and functional regulation. Accurate determination of the C-terminal sequence is essential for understanding these biological functions. Traditional methods, such as carboxypeptidase method, have limitations for C-terminal sequencing. In contrast, LC-MS/MS technology provides higher sensitivity and resolution, overcoming these limitations.

 

Sample Preparation

The first step in C-terminal sequencing is sample preparation, typically involving protein extraction, purification, and digestion. To ensure accurate C-terminal sequencing, highly specific enzymes like trypsin are commonly used. After trypsin digestion, the protein sample generates a series of peptides, with the C-terminal peptide identified by LC-MS/MS.

 

Liquid Chromatography (LC) Separation

In LC-MS/MS analysis, liquid chromatography (LC) separates complex peptide mixtures. Reverse-phase high-performance liquid chromatography (RP-HPLC) is the most common method, utilizing the different retention times of peptides on a hydrophobic column. Gradient elution effectively separates peptides for subsequent mass spectrometry analysis.

 

Mass Spectrometry (MS) Identification

Mass spectrometry is the core step in C-terminal sequencing. Common techniques include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In ESI-MS/MS, separated peptides are ionized and introduced into the mass spectrometer. Collision-induced dissociation (CID) generates fragment ions. Analyzing the mass-to-charge ratio (m/z) of these fragment ions reconstructs the peptide sequence information.

 

Data Analysis

The final step in C-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 C-terminal sequence.

 

LC-MS/MS technology provides an efficient and accurate method for C-terminal sequencing. Its high sensitivity and resolution enable the identification of even low-abundance C-terminal peptides. As mass spectrometry and data analysis methods advance, the application of LC-MS/MS in proteomics research will become more widespread and in-depth. MtoZ Biolabs provides integrate C-Terminal sequencing service.