iTRAQ Analysis of Proteomics
Proteomic iTRAQ analysis refers to a technique that utilizes both iTRAQ labeling technology and mass spectrometry for protein analysis, primarily used in quantitative proteomics analysis. Biotech company BGI provides mass spectrometry-based protein iTRAQ analysis services.
Proteomic iTRAQ Analysis
Proteomic iTRAQ analysis refers to a technique combining iTRAQ labeling and mass spectrometry for protein analysis. iTRAQ (Isobaric tag for relative and absolute quantitation) is a peptide in vitro labeling technology developed by AB SCIEX. In quantitative proteomics, iTRAQ employs multiple isotopic tags (4 or 8) to label peptide fragments from different sources in a single experiment at the N-terminus. These fragments are then analyzed by tandem mass spectrometry, allowing for the simultaneous comparison of the relative and absolute abundance of proteins from different sources.
Principle of Proteomic iTRAQ Analysis
An iTRAQ tag consists of three parts: a reporter group, a balance group, and a peptide reactive group. The reporter and balance groups together form an isobaric tag. The reporter group's weight varies among different tags and indicates the abundance level of the protein sample. The balance group balances the mass difference of the reporter group, ensuring that the isobaric tag weight is consistent, which guarantees that the m/z of the labeled peptide fragment remains the same. The peptide reactive group covalently binds to the N-terminus of peptides and the amino group of lysine side chains, thus labeling the peptide fragment.
In proteomic iTRAQ analysis, proteins are first cleaved into peptide fragments and then differentially labeled with iTRAQ reagents. Since iTRAQ reagents are isobaric, the molecular weight of the same labeled peptide is identical in the first level of mass spectrometry detection, necessitating a second level of mass spectrometry analysis. During the secondary mass spectrometry analysis, the bonds between the reporter group, balance group, and peptide reactive group are broken, with the balance group being lost. The difference in ion intensity of different reporter groups represents the relative abundance of the peptide they label. Concurrently, the amide bonds within the peptide are broken, generating fragment ions whose masses can be identified by database searching and comparison, leading to the identification of corresponding proteins.
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