Quantitative Proteomics Analysis Using iTRAQ/TMT

Quantitative proteomics is an essential technology in biomedical research, providing detailed information on protein expression levels within cells. iTRAQ (isobaric tags for relative and absolute quantitation) and TMT (Tandem Mass Tag) are two widely used tagging techniques that enable relative and absolute quantification of multiple samples. These techniques allow researchers to identify and quantify thousands of proteins in complex biological samples, offering new insights into disease mechanisms, drug development, and biomarker discovery.

 

iTRAQ and TMT are both tag-based methods based on mass spectrometry analysis. They label peptide segments from different samples, allowing simultaneous measurement in a single mass spectrometry run. iTRAQ uses four different labels (114, 115, 116, 117), while TMT offers various labels (such as TMT6, TMT10) suitable for analysis of different numbers of samples. After labeling, the peptide segments release specific reporter ions during mass spectrometry analysis, and researchers can calculate the relative abundance of target proteins in each sample using the relative abundance of these reporter ions.

 

Experimental Steps

1. Sample Preparation

Sample preparation is a critical step for successful iTRAQ/TMT analysis. First, proteins need to be extracted from biological samples (such as cells, tissues, or biological fluids).

 

2. Digestion

The extracted proteins are typically digested using enzymes like trypsin to convert them into shorter peptide segments. This digestion process not only enhances the efficiency of mass spectrometry analysis but also improves the ionization efficiency of the peptides.

 

3. Tagging

The digested peptides react with iTRAQ or TMT reagents for tagging. This step must be performed under appropriate conditions to ensure complete and specific binding of the tags to the peptides.

 

4. Sample Mixing

The tagged peptides should be mixed according to the experimental design, usually combining the peptides from different samples in the same proportion to enhance the accuracy of the analysis.

 

5. Mass Spectrometry Analysis

The mixed peptides are then analyzed using a mass spectrometer. The mass spectrometer separates and detects the peptides based on their mass-to-charge ratio (m/z), generating a mass spectrum.

 

6. Data Analysis

The generated mass spectrometry data must be analyzed using software to extract the abundance information of the reporter ions. Quantitative results can be obtained by comparing the relative abundances, enabling further analysis of protein expression changes.

 

Applications

1. Disease Mechanism Research

By comparing protein expression differences between healthy and diseased samples, revealing molecular mechanisms of diseases.

 

2. Biomarker Discovery

Identifying proteins associated with diseases as potential biomarkers for early diagnosis or efficacy assessment.

 

3. Drug Development

Evaluating changes in protein expression after drug treatment to help understand the drug's mechanism of action.

 

Using iTRAQ/TMT for quantitative proteomics analysis has unique advantages and limitations. The main advantages include high throughput, high sensitivity, and the ability to simultaneously quantify various samples. However, these techniques also have certain limitations, such as high costs, stringent sample processing requirements, and the complexity of data analysis.