Workflows of iTRAQ, TMT, and SILAC in Protein Quantification

Protein quantification is crucial for understanding cellular functions, disease mechanisms, and biological system complexities in proteomics research. iTRAQ (Isobaric Tags for Relative and Absolute Quantification), TMT (Tandem Mass Tag), and SILAC (Stable Isotope Labeling by Amino acids in Cell culture) are three widely used quantitative proteomics methods. Each method has unique advantages and is extensively applied in relative or absolute protein quantification.

 

SILAC Workflow

SILAC is a stable isotope labeling method used in live-cell experiments. Its principle involves the incorporation of stable isotope-labeled amino acids into the proteins synthesized by cells, facilitated by adding labeled amino acids to the culture medium. The workflow includes the following steps:

 

1. Cell Culture

Two groups of cells are cultured in media containing either labeled amino acids (e.g., ^13C6-L-lysine) or unlabeled amino acids.

 

2. Protein Extraction

Once the culture is complete, cells are harvested, and total protein is extracted.

 

3. Protein Digestion

The extracted proteins are enzymatically digested, typically using trypsin, to generate peptides.

 

4. Peptide Mixing

Peptides from the two cell groups are mixed in a fixed ratio, allowing comparison of protein abundance between the groups in a single mass spectrometry analysis.

 

5. Mass Spectrometry Analysis

The mixed peptides are analyzed by mass spectrometry, and the relative protein abundance is determined by comparing the signal intensities of labeled and unlabeled peptides.

 

iTRAQ Workflow

iTRAQ is a chemical labeling method for relative protein quantification, often used to analyze multiple samples simultaneously. The iTRAQ workflow involves the following:

 

1. Sample Preparation and Protein Extraction

Total proteins are extracted from multiple samples.

 

2. Protein Digestion

Protein samples are digested into peptides, typically using trypsin.

 

3. Peptide Labeling

The peptides are labeled with iTRAQ tags, with each sample receiving a different isotopic tag. These tags have identical masses but generate unique reporter ions during mass spectrometry fragmentation.

 

4. Peptide Mixing

The labeled peptides from different samples are mixed for simultaneous mass spectrometry analysis.

 

5. Mass Spectrometry Analysis

Peptides are separated and analyzed by liquid chromatography coupled with mass spectrometry (LC-MS/MS). The resulting reporter ions provide relative quantification of proteins across samples.

 

TMT Workflow

TMT, similar to iTRAQ, is also a labeling technique for protein quantification. The TMT workflow involves:

 

1. Sample Preparation and Protein Extraction

Proteins are extracted from multiple experimental groups.

 

2. Protein Digestion

Proteins are enzymatically digested into peptides.

 

3. TMT Labeling

Peptides from each group are labeled with TMT tags. Although the tags have the same mass, they produce distinct ion signals during mass spectrometry analysis.

 

4. Peptide Mixing

The labeled peptides are mixed and analyzed together.

 

5. Mass Spectrometry Analysis

LC-MS/MS is used for peptide separation and analysis. The relative abundance of proteins is calculated based on the signal intensities of the reporter ions generated.

 

iTRAQ, TMT, and SILAC are key technologies for protein quantification. SILAC is well-suited for live-cell experiments with direct incorporation of labeled amino acids, while iTRAQ and TMT use chemical labels to compare multiple samples. Whether used for relative or absolute quantification, these methods rely on high-resolution mass spectrometry coupled with liquid chromatography to separate peptides, providing accurate and comprehensive quantification of proteins.