Procedure for Peptide Sequencing Based on De Novo Method

In proteomics research, peptide sequencing is a crucial technique for understanding protein structure and function. Unlike traditional database search methods, De Novo sequencing does not rely on known protein sequence databases but directly derives amino acid sequences from mass spectrometry data.

 

Sample Preparation

Sample preparation is the first step in peptide sequencing. It typically includes protein extraction, purification, and enzymatic digestion. Enzymes like trypsin are commonly used to break down proteins into smaller peptides, which facilitate subsequent analysis.

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the core step of peptide sequencing. Using mass spectrometers (such as MALDI-TOF or ESI-MS), the mass-to-charge ratio (m/z) of peptide fragments is measured, generating a mass spectrum. The mass spectrum displays the m/z values of different peptide fragments along with their corresponding intensities.

 

Data Processing

Mass spectrometry data processing involves converting raw data into usable information. Steps include noise reduction, peak detection, and mass calibration. These steps are crucial to ensure the accuracy and reliability of the data.

 

Sequence Deduction

Sequence deduction is the critical step in De Novo peptide sequencing. Algorithms like PepNovo and Novor are commonly used. These algorithms analyze the intensity and position of peaks in the mass spectrum to deduce possible peptide sequences.

 

Sequence Validation

To validate the deduced peptide sequences, comparison with known sequence databases is essential to ensure accuracy. Additionally, methods such as tandem mass spectrometry (MS/MS) provide extra information to assist in validation.

 

Data Interpretation and Applications

The final peptide sequences can be utilized in various biological studies, such as protein function analysis and protein-protein interaction studies. De Novo sequencing is particularly valuable in discovering new proteins and detecting mutations.