Part I: Detailed Description of iTRAQ/TMT Tag Structure and Relative Quantification Principle
Introduction to iTRAQ and TMT Tag
Speaking of iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag), many of us may think that they are two different quantitative proteomics techniques, but in fact, except the differences in labeling specifications and label molecular structure as well as developer, the principle of their labeling peptides are basically the same. iTRAQ was firstly developed by AB SCIEX, followed by whcih TMT was developed by Thermo Fisher. Although they have different patents, they use almost the same principle.
The iTRAQ and TMT marker are essentially a chemical in vitro labeling reagent that specifically labels peptides produced by protein digestion. The iTRAQ and TMT-labeled protein samples are available in different sizes. In contrast, TMT can label a wider range of samples and simultaneously quantify multiple sets of 2 protein samples at least and 10 at most. By labeling multiple sets of different samples, TMT and iTRAQ can simultaneously compare protein levels in normal tissue samples and tumor tissue samples, as well as accurately detect changes in protein levels at different stages of tumor development.
Although iTRAQ and TMT have the same labeling principle, there are differences in the structure of these two tags.
Comparison Between iTRAQ Molecular Structure and TMT Molecular Structure
Introduction to iTRAQ and TMT Tag
Speaking of iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag), many of us may think that they are two different quantitative proteomics techniques, but in fact, except the differences in labeling specifications and label molecular structure as well as developer, the principle of their labeling peptides are basically the same. iTRAQ was firstly developed by AB SCIEX, followed by whcih TMT was developed by Thermo Fisher. Although they have different patents, they use almost the same principle.
The iTRAQ and TMT marker are essentially a chemical in vitro labeling reagent that specifically labels peptides produced by protein digestion. The iTRAQ and TMT-labeled protein samples are available in different sizes. In contrast, TMT can label a wider range of samples and simultaneously quantify multiple sets of 2 protein samples at least and 10 at most. By labeling multiple sets of different samples, TMT and iTRAQ can simultaneously compare protein levels in normal tissue samples and tumor tissue samples, as well as accurately detect changes in protein levels at different stages of tumor development.
Although iTRAQ and TMT have the same labeling principle, there are differences in the structure of these two tags.
Comparison Between iTRAQ Molecular Structure and TMT Molecular Structure
From the above figure, we can see that iTRAQ and TMT tags have obvious differences in structure, but they do have something in common. Firstly, both tags are composed of reporter groups, balance groups and peptide reactive groups. The total weight of the respective reporter and balance groups of the labels is constant. In addition, the peptide reaction group structure of the two tags is also the same. The difference between iTRAQ and TMT tags lies in the structure of the equilibrium group. It can be seen from the figure below that the equilibrium group structure of TMT is more complicated than that of iTRAQ tags. The equilibrium group of iTRAQ has only dozens of Da, while the equilibrium group of TMT is close to 200 Da, which is also the cause of the difference in their mass.
After a brief introduction to the structure of the two tags, let’s take the iTRAQ 4-plex tag as an example to learn more about the structure of the iTRAQ molecular tag.
iTRAQ Molecular Structure
The iTRAQ tag molecular backbone is composed of three core components, a reporter group, a balance group, and an amine-specific reactive group. The mass of the reporter groups of different iTRAQ tags is different. The reporter group mass of 4-plex tags is 114 Da, 115 Da, 116 Da and 117 Da respectively. The balance group mass of 4-plex tags is 31 Da, 30 Da, 29 Da and 28 Da respectively, which makes the total reporter group mass of the 4 iTRAQ tags all equals to 145 Da. Another core component is the amine-specific reactive group, whose main function is to undergo a displacement reaction with the free n terminal amino group of the polypeptide, so that the isotope tag is covalently cross-linked to the N-terminus of the peptide. As mentioned above, the reporter group of the label differs only by a few Da. How does it happen? Actually, it uses the principle of isotope labeling.
