iTRAQ Sequencing Uncovered: From Technique to Application

iTRAQ sequencing is a commonly used protein quantification mass spectrometry technique. It labels amino acid residues in protein samples to compare protein expression levels between different samples. This article will comprehensively analyze the iTRAQ sequencing technology from the aspects of technical principles, experimental steps, and application cases.

 

Technical Principles

The core principle of iTRAQ sequencing is to use chemical labelling methods to label proteins in different samples, and then analyze them with a mass spectrometer to quantitatively compare protein expression levels in different samples. The specific steps are as follows:

 

1. Sample Preparation

Extract and digest proteins from the samples to be tested to obtain protein fragments.

 

2. Labelling Reaction

Use iTRAQ reagents to label protein fragments in different samples. iTRAQ reagents are compounds containing isotope labels, and different iTRAQ reagents correspond to different samples. The labelling reaction will bind the iTRAQ reagent to the amino group in the protein fragment.

 

3. Sample Mixing

Mix the labelled samples to mix protein fragments from different samples together.

 

4. Mass Spectrometry Analysis

Analyze the mixed sample by mass spectrometry to obtain the mass spectrum of the protein fragment.

 

5. Data Analysis

By processing and analyzing the mass spectrometry data, the relative expression levels of proteins in different samples can be obtained.

 

Experimental Steps

The experimental steps of iTRAQ sequencing mainly include sample preparation, labelling reaction, sample mixing, mass spectrometry analysis, and data analysis. The following will detail the operation points of each step:

 

1. Sample Preparation

Select appropriate samples, extract and digest proteins. During the extraction process, it is necessary to use appropriate buffer and protease to ensure the integrity and digestion effect of the protein.

 

2. Labelling Reaction

According to the experimental design, select the appropriate iTRAQ reagent for the labelling reaction. The reaction time and temperature need to be controlled according to the requirements of the reagent to ensure the effect of labelling.

 

3. Sample Mixing

Mix the labelled samples according to the ratio of the experimental design. It is necessary to stir well during the mixing process to ensure that the protein fragments from different samples are mixed evenly.

 

4. Mass Spectrometry Analysis

Analyze the mixed samples by mass spectrometry. You can choose liquid chromatography-mass spectrometry technology or mass spectrometry imaging technology for analysis. The parameter setting of the mass spectrometer needs to be optimized according to the characteristics of the sample to obtain high-quality mass spectrometry data.

 

5. Data Analysis

By processing and analyzing the mass spectrometry data, the relative expression levels of proteins can be obtained. Common data analysis methods include comparative group difference analysis, cluster analysis, and pathway analysis.

 

Application

iTRAQ sequencing technology is widely used in biomedical research and drug development. Here are a few typical application cases:

 

1. Cancer Research

By comparing the differences in protein expression levels between cancer cells and normal cells, potential targets and signaling pathways related to cancer can be discovered, providing new ideas for the diagnosis and treatment of cancer.

 

2. Drug Screening

iTRAQ sequencing technology can be used to study the mechanism of action of drugs, and screen potential therapeutic targets and candidate drugs.

 

3. Discovery of Biological Markers

By comparing the differences in protein expression in different disease states, biological markers related to the disease can be discovered, providing a basis for early diagnosis and treatment of the disease.

 

4. Research on Protein Interactions

iTRAQ sequencing technology can be used to analyze the protein interaction network, revealing the mode and mechanism of protein interaction.

 

As a protein quantification mass spectrometry technique, iTRAQ sequencing technology has the characteristics of high throughput, high sensitivity, and high precision. It is widely used in biomedical research and drug development. By quantitatively comparing proteins in different samples, differences in protein expression can be revealed, providing new ideas and methods for disease diagnosis and treatment. With the continuous development and improvement of technology, iTRAQ sequencing technology will play an increasingly important role in the field of life sciences.