Workflow of N-Terminal Sequencing

Sample Preparation

The initial phase of N-terminal sequencing involves rigorous sample preparation. This process typically requires protein extraction, purification, and concentration to ensure that the protein samples are sufficiently pure and concentrated. Techniques commonly employed include centrifugation, gel filtration, and affinity chromatography, each chosen based on the specific requirements of the sample.

 

Protein Digestion

Following sample preparation, proteins are digested enzymatically to produce peptides suitable for analysis. Trypsin is preferred for its high specificity, cleaving at each arginine or lysine residue, thereby preserving the integrity of the N-terminal information.

 

Peptide Separation

The peptides generated from digestion are then separated using high-performance liquid chromatography (HPLC). This critical step directly impacts the efficiency and accuracy of the subsequent mass spectrometry analysis. Optimal chromatographic conditions are crucial for enhancing the purity and recovery rates of the targeted peptides.

 

Sequence Determination

The isolated peptides undergo sequencing through advanced techniques like electrospray ionization-mass spectrometry/mass spectrometry (ESI-MS/MS). This method provides precise determinations of peptide masses and their sequential arrangements. Peptides are first ionized, then further fragmented in the mass spectrometer, which allows for a detailed analysis of their amino acid compositions.

 

Data Analysis

The concluding step involves sophisticated data analysis, where software tools such as Mascot or Sequest are employed to compare mass spectrometry results with protein databases. This comparison helps in accurately determining the N-terminal sequences. This phase is technically demanding, requiring intricate adjustments and interpretations to align experimental data with known sequences.

 

The workflow of N-terminal sequencing covers a spectrum of critical steps, from sample preparation to intricate data analysis. MtoZ Biolabs provides integrate N-Terminal sequencing service.