iTRAQ Quantitative Proteomic Technique

The Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) technology is a type of multiplexed peptide in vitro labeling technology developed by AB Sciex. This technology uses 4 or 8 isotope tags to label the amino groups of peptides specifically, followed by tandem mass spectrometry analysis, allowing for simultaneous comparison of relative or absolute protein quantities in 4 or 8 different samples.

 

Services at Mtoz Biolabs

iTRAQ reagents consist of three components: reporting group, balancing group, and peptide reactive group, forming 4 or 8 isotope labels with equal relative molecular mass (145 or 305).

 

1. Reporting Group

Indicates the abundance level of protein samples. The masses of the 4-plex label reporting groups are 114 Da, 115 Da, 116 Da, and 117 Da.

 

2. Balancing Group

Balances the mass difference of the reporting group, making the mass of isobaric labels consistent and ensuring the same peptide segment m/z. The masses of the 4-plex label balancing groups are 31 Da, 30 Da, 29 Da, and 28 Da, making the total mass of the 4 iTRAQ labels equal to 145 Da.

 

3. Peptide Reactive Group

It can undergo substitution reactions with the side chain of lysine or the N-terminus of the peptide segment, completing the labeling of the peptide segment.

 

Analysis Workflow

1. Protein Digestion

The protein samples are digested into peptides.

 

2. iTRAQ Labeling

Different isotope tags are used to label different peptide samples.

 

3. Sample Mixing

The labeled peptide segments from different groups are mixed in equal amounts, then subjected to vacuum freeze-drying to remove excess solvents and reagents.

 

4. LC-MS/MS Spectrometry Detection and Analysis

The mass spectrometer is used to perform tandem mass spectrometry analysis on the mixed labeled peptide segments. In the mass spectrometer, the peptide segments are fragmented into smaller pieces and produce ions with different masses. The intensity differences of these ions represent the relative abundance of different peptide segments.

 

5. Data Analysis

Software is used to process and analyze the raw spectrometry data. After iTRAQ labeling, the same peptide segment from different samples has the same molecular weight, so it appears as a single peak in the primary spectrometry diagram. After secondary fragmentation, the bonds between the reporting group, balancing group, and peptide reactive group break, producing reporting ions with specific masses. By comparing the peak height and area of these reporting ions, the expression quantity of the same peptide segment among different samples can be quantitatively analyzed.

 

Apart from the difference in the number of samples labeled, iTRAQ is largely similar to TMT in other aspects (principle and application). Both possess the characteristics of high throughput and high accuracy and are widely used in quantitative analysis and large-scale comparative proteomics research. MtoZ Biolabs uses Thermo Fisher's Q ExactiveHF mass spectrometry platform, Orbitrap Fusion mass spectrometry platform, Orbitrap Fusion Lumos mass spectrometry platform combined with Nano-LC, provides iTRAQ/TMT based in vitro quantitative proteomics analysis service. In addition, there is also the in vivo labeling SILAC technology suitable for different samples and scenarios, which can meet your different needs, welcome to consult.