Mechanism of iTRAQ/TMT and MultiNotch Labeling in Proteomics

In modern life science research, quantitative proteomics has become a key tool for understanding the complexity of cellular biology. iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tags) are two widely used isotope labeling-based quantitative proteomics technologies. These technologies introduce mass-differentiated tags into samples, enabling precise relative and absolute quantification across multiple samples simultaneously.

 

Labeling Principles of iTRAQ/TMT

The working principles of iTRAQ and TMT revolve around isotope labeling. These labels are small, chemically synthesized molecules that covalently bind to specific sites on proteins or peptides, typically the amino termini or lysine residues. Each labeling group comprises a reporter ion, a balance region, and a reactive group. During mass spectrometry analysis, these labels generate characteristic fragment ions, which serve as indicators for quantitative analysis.

 

The core strength of iTRAQ and TMT technologies lies in the chemical equivalence of their labels. Although these labels are chemically identical, they introduce subtle mass differences by incorporating varying numbers of isotopes, such as 13C, 15N, or 18O. This difference allows for the discrimination of labels in a mass spectrometer. Importantly, despite these mass differences, the labels behave identically in all other respects—such as during electrophoresis or liquid chromatography—prior to mass spectrometry analysis. This feature makes iTRAQ/TMT technologies particularly advantageous for the simultaneous analysis of multiple samples.

 

Sample Preparation and Labeling Process

The iTRAQ/TMT analysis workflow typically involves the following steps:

 

1. Protein Extraction

Total proteins are extracted from the sample, ensuring high quality and purity for downstream analysis.

 

2. Protein Digestion

Extracted proteins are digested into peptides using trypsin or another suitable protease.

 

3. Labeling Reaction

iTRAQ or TMT reagents are then added to the peptide samples, resulting in covalent binding to the amino termini or lysine residues. Different tags are used for different samples, allowing for their differentiation during mass spectrometry analysis.

 

4. Sample Mixing

The labeled peptide samples are combined into a single mixture containing multiple labels.

 

5. Separation and Analysis

The mixed samples are separated by liquid chromatography, followed by mass spectrometry analysis to obtain quantitative data.

 

Mass Spectrometry Analysis and Data Processing

In the mass spectrometry analysis phase, iTRAQ- and TMT-labeled peptides are initially detected by MS1, which measures their mass-to-charge ratios. Subsequently, these peptides are selectively fragmented to produce MS2 spectra. In the MS2 spectra, each labeled peptide generates characteristic reporter ions, whose intensities correlate with the relative abundance of the protein in the respective sample.

 

Data processing is a crucial component of iTRAQ/TMT analysis. By comparing the intensities of reporter ions across different samples, researchers can calculate the relative expression levels of target proteins. Moreover, absolute protein concentrations can be determined by incorporating absolute quantification standards. This level of quantification is vital for comprehensively understanding protein functions and their underlying mechanisms.

 

iTRAQ and TMT technologies offer powerful solutions for the parallel analysis of multiple samples, greatly enhancing the efficiency and accuracy of quantitative proteomics. However, despite their advantages, these technologies require high-cost instrumentation and involve complex data processing workflows, which may limit their accessibility in certain research settings.