iTRAQ: A Proteomics Research Tool

Posted by Dora West on December 28th, 2021

Proteomics is a combination of two words: protein and genomics. Proteomics studies all the proteins expressed by a cell or even an organism. Essentially, it is the study of proteins and their characteristics on a large scale, including protein expression levels, post-translational modifications, and protein-protein interactions. Various relevant information can be obtained for an overall and comprehensive understanding of cell metabolism and other processes. In other words, proteomics is a science that explores disease mechanisms, cell models, and functional connections at the protein level based on comprehensive protein properties research.

With the rapid development of proteomics in recent years, its corresponding methodological research has also made considerable progress. A series of new technologies have been integrated into the research of proteomics, which has greatly promoted the development of this subject. iTRAQ technology combined with highly sensitive and accurate tandem mass spectrometry or/and multidimensional liquid chromatography has become one of the main tools for protein qualitative and quantitative research.

Principles of iTRAQ technology

iTRAQ is an in vitro isobaric isotope-labeled relative and absolute quantitative technology developed by AB SCIEX. This technology uses 8 (or 4) different isotope reagents to simultaneously label and compare 8 (or 4) different protein samples. These reagents consist of 3 different chemical tags: a reporting part, a peptide reaction part, and a balance part.

1. In the reporting part, customers can choose the number of marks as needed.
2. The peptide reactive group connects the iTRAQ tag with the N-terminal group of the peptide and each lysine side chain, which can label all the enzymatically digested peptides.
3. The balance position can ensure that the same peptides of different samples labeled with iTRAQ reagent have the same mass-to-charge ratio.

Therefore, if any iTRAQ reagent is changed, the molecular weights of different isotopes are exactly the same in the first-stage mass spectrometry after labeling the same polypeptide. In tandem mass spectrometry, the balance group of the isobaric element-labeled peptide is neutrally lost in the secondary mass spectrometry. Signal ions appear as peaks with different mass-to-charge ratios. Therefore, based on the height and area of the peak, quantitative information about the protein can be obtained.

Features of iTRAQ technology

A commonly used technique for quantifying labeled proteins, iTRAQ technology has many advantages:

1. In the same experiment, iTRAQ can quantitatively compare up to 8 different samples at the same time. It minimizes the influence of system errors such as ion suppression effects, background noise, and instrument conditions.

2. iTRAQ technology can label almost all proteins in the sample. At the same time, qualitative and quantitative studies can be performed on phosphorylated proteins, glycosylated proteins and other post-translationally modified proteins, from which more detailed information can be obtained.

3. The labeling process is simple in that it can be completed in 1 h at room temperature, and the mass spectrometry detection is sensitive. Therefore, iTRAQ technology can provide quantitative information for sample analysis, membrane protein research, discovery, and identification of disease markers in different time periods, and can perform absolute quantification for the target protein of interest.