Workflow of Peptide Sequencing by Mass Spectrometry

Mass spectrometry (MS) sequencing is a powerful tool widely used in proteomics research to analyze and identify the amino acid sequences of proteins.

 

Sample Preparation

The first step in mass spectrometry sequencing is sample preparation. Typically, samples are complex mixtures of proteins requiring proper processing for subsequent analysis. The main steps include:

 

1. Protein Extraction

Proteins are extracted from cells, tissues, or biological fluids.

 

2. Protein Purification

Impurities are removed through centrifugation, filtration, or chromatography, resulting in purified protein samples.

 

3. Protein Digestion

Enzymes such as trypsin are used to cleave proteins into smaller peptide fragments, which are more suitable for mass spectrometry analysis.

 

Peptide Separation

After digestion, peptides need to be separated. Liquid chromatography (LC) is a commonly used method. LC separates peptides based on characteristics like hydrophobicity, size, and polarity, thus reducing the complexity for mass spectrometry analysis.

 

Mass Spectrometry Analysis

1. Ionization

Peptides are ionized and introduced into the mass spectrometer, commonly using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).

 

2. Mass Analysis

The ionized peptides are separated and detected based on their mass-to-charge ratio (m/z). Common mass spectrometers include time-of-flight (TOF), quadrupole, and ion trap mass spectrometers.

 

Data Acquisition and Processing

The mass spectrometer generates extensive data that must be processed to obtain peptide mass-to-charge ratio information. Data processing steps include:

 

1. Peak Detection

Identifying ion peaks in the mass spectrum.

 

2. Peak Matching

Comparing detected peaks with known peptide fragments in databases.

 

3. Sequence Inference

Inferring the peptide amino acid sequences from the mass spectrometry data.

 

Database Search and Identification

Peptide sequences are identified through database searches using the mass spectrometry data. Common databases include NCBI and UniProt. Search algorithms compare experimental mass spectra with theoretical spectra in databases to find the best matches.

 

Result Verification and Analysis

1. Repeat Experiments

Ensuring result reliability through repeated experiments.

 

2. Bioinformatics Analysis

Further analyzing peptide functions and interactions using bioinformatics tools.

 

MtoZ Biolabs provides integrate peptide sequencing service by mass spectrometry.