Detailed Explanation of iTRAQ/TMT Tag Structure and Relative Quantification Principles
iTRAQIntroduction to iTRAQ and TMT Labels
When it comes to iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag), many people might think they are two different quantitative proteomics technologies. However, apart from differences in labeling specifications, molecular structure, and developers, the principle of labeling peptides is essentially the same for both. iTRAQ was first developed by AB SCIEX, followed by TMT, which was developed by Thermo Fisher. Despite having different patents, they use almost the same principle.
iTRAQ and TMT markers are essentially chemical in vitro labeling reagents specifically for labeling peptides derived from protein digestion. The protein samples labeled with iTRAQ and TMT have different sizes. In contrast, TMT can label a wider range of samples and quantify multiple groups of protein samples simultaneously, at least 2 and up to 10. By labeling multiple sets of different samples, TMT and iTRAQ can simultaneously compare protein levels in normal tissue samples and tumor tissue samples and accurately detect changes in protein levels at different stages of tumor development.
Although the labeling principles of iTRAQ and TMT are the same, there are differences in their label structures.
iTRAQComparison of Molecular Structures with TMT
From the above figure, it is evident that there are distinct structural differences between iTRAQ and TMT labels, but they do share some commonalities. Firstly, both labels consist of a reporter group, a balance group, and a peptide reactive group. The total weight of their respective reporter and balance groups is constant. Additionally, the peptide reactive group structure of both labels is also identical. The difference between iTRAQ and TMT labels lies in the structure of the balance group. As shown in the figure, the balance group structure of TMT is more complex than that of the iTRAQ label. The balance group of iTRAQ is only a few dozen Daltons, while that of TMT is nearly 200 Daltons, which is the reason for their different masses.
After briefly introducing the structure of the two labels, let's take the iTRAQ 4-plex label as an example to further understand the structure of the iTRAQ molecular label.
iTRAQMolecular Structure
The iTRAQ label molecular framework consists of three core parts: a reporter group, a balance group, and a peptide reactive group. The mass of the reporter group varies among different iTRAQ labels. For the 4-plex label, the reporter group masses are 114 Da, 115 Da, 116 Da, and 117 Da, respectively. The balance group masses for the 4-plex label are 31 Da, 30 Da, 29 Da, and 28 Da, which ensures that the total mass of the four iTRAQ labels is equal at 145 Da. Another core component is the peptide reactive group, whose main role is to undergo a substitution reaction with the free N-terminal amino group of the peptide, allowing the isotope label to covalently cross-link with the N-terminal of the peptide.
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