Part II: Detailed Description of iTRAQ/TMT Tag Structure and Relative Quantification Principle


As shown below, the reporter group whose mass is 114 contains one C13, and the mass of the reporter group is increased by 1 Da. At the same time, the balance group contains one C13 and one O18, and the total mass is increased by 3 Da, so that the total mass of the 114 label is increased by 4 Da. By analogy, the reporter group of 115 increases by 2 Da, the balance group will increase by 2 Da. The reporter group of 116 increases by 3 Da, the balance group will increase by 1 Da. The reporter group of 117 increases by 4Da, the balance group will increase by 0 Da. After the above four tags are labeled with different isotopes for its reporter group and balance group, the respective reporter group and balance group have different masses. However, as the total mass added is the same, the total mass of the label equals.

iTRAQ Relative Quantitative Research Principle

The protein is first cleaved into peptides and then differentially labeled with iTRAQ reagent. Since the iTRAQ reagents are equivalent, different isotopes are detected with MS after labeling the same polypeptide, and the molecular weight is completely the same. The precursor ions detected with MS are subjected to collision-induced dissociation by tandem mass spectrometry, and the product ions are analyzed by MS/MS. During the analysis of MS/MS, the bonds between the reporter group, the balance group, and the amine-specific reactive group are broken, and the reporter group is lost, resulting in a low mass to charge ratio (m/z) of reporter ions. As MS/MS can analyze reporter group with a relative molecular mass difference of 1, the difference in ionic strength of different reporter groups represents the relative abundance of the polypeptides it labels. At the same time, amide bond cleavage in polypeptide forms a series of b ions and y ions, and the mass of the ion fragments is obtained. The corresponding protein precursors can be identified by database query and comparison.


iTRAQ Relative Quantitative Research Processes

lDigest different protein samples into peptide fragments.
lLabel the polypeptide fragments produced by digestion of different protein samples separately using different iTRAQ tags, but the total mass of the different iTRAQ tags is the same.
lProportional mixing of each labeled sample
lThe labeled peptide fragments are separated by MS. In MS, the iTRAQ tags cannot be distinguished by the same peptides from different protein samples due to the same total mass, so they will enter the MS/MS together.
l In MS/MS, the balance group of iTRAQ will be broken under high energy collision, so the reporter group is released. The peptide is also released, and the collision is broken into secondary fragments. By analyzing the amino acid sequence of the peptide, the corresponding protein sequence can be inferred. At the same time, the signal intensity of each reporter group in the mass spectrum represents the relative abundance of the polypeptide in the four groups of samples, that is, the relative expression levels of the corresponding proteins in the four groups of samples are presented.







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