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.







Comments

Post a Comment

Popular posts from this blog

Practice of N-terminal Sequencing by Edman Degradation Technology in Protein and Peptide Sequence Analysis

Image
The N-terminal amino acid sequence of protein is one of the key quality attributes of biological drugs. N-terminal sequencing by Edman degradation  is a routine method to analyze this property. The analysis of 15 amino acid sequences on the N-terminal by Edman degradation method is usually a necessary item when applying for biological drugs . At  the same time, it is also an annual inspection item of many listed biological drugs (see Pharmacopoeia 2015). In scientific research, N-terminal sequencing can be used to provide key information for the confirmation of unknown or uncertain theoretical sequences. So this method is widely used in scientific research and industry.  W hat are its principles ? H ow to analyze the results ? Advice for sample delivery? P rinciples In short, we can understand it from these two aspects: The first step  is Edman degradation . T hrough the reaction of PITC (Phenylisothiocyanate), an organic reagent, and α - amino acid at t...
Read more

De Novo Protein Sequencing Procedures and Features

Image
The sequence of a protein molecule is the basis for the function of a protein. Accurate determinat ion of protein sequence plays an important role in the study of protein functional epitopes, detection of commercial monoclonal antibodies, vaccines, and kits. Although there are many studies on protein sequencing, accurate sequencing of proteins is still not as fast and accurate as that of DNA. Protein sequencing is mainly hampered by the purification of protein samples, and the complexity of factors that make up the amino acid com position of proteins. De novo   protein sequencing  is  also called novel protein sequencing. This technique deduces t he amino acid sequence based on the difference in mass between a series of regular fragment ions generated by the peptide collision with an inert gas. We can guess the amino acid sequence and post-translational modifications based on the y ion and b ion at the cleavage of the peptide bond. De novo  sequencing has a uniq...
Read more

Part I: Basic Knowledge of Quantitative Analysis of Protein Acetylation Modification

Image
After the protein is translated in the cell, it is processed through a very important step before being transported to the corresponding organelle and undergoing specific biological effects. The role of protein   post-translational modification   ( PTM ) is primarily to alter the activity, localization or function of the protein. Protein PTM  also further increases the diversity and complexity of cellular pathway mechanisms and life activities. Except for acetylation , c ommon protein PTM  include s   phosphorylation , glycosylation , ubiquitination , and the like. Protein  A cetylation  M odification Protein acetylation modification, as the name suggests, refers to the grafting of acetylated groups on the original basis of the protein. In cells, the acetylation modification reaction is catalyzed by an acetyltransferase, and the acetyl group of acetyl-CoA is transferred and added to the protein lysine residue. In earlier studies, acetylati...
Read more