Isobaric tag for relative and absolute quantitation
Isobaric tags for relative and absolute quantitation (iTRAQ) are a non-gel-based technique used to quantify proteins from different sources in a single experiment. It uses isotope-coded covalent tags. iTRAQ is used in proteomics to study quantitative changes in the proteome.[1][2][3]
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[edit] Procedure
The method is based on the covalent labeling of the N-terminus and sidechain amines of peptides from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS). A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.
[edit] Data evaluation
[edit] Peptide level
The signals of the reporter ions of each MS/MS spectrum allow for calculating the relative abundance (ratio) of the peptide(s) identified by this spectrum. The abundance of the reporter ions may consist of more than one single signal in the MS/MS data and the signals have to be integrated in some way from the histogram spectrum.
[edit] Protein level
The combined ratios a proteins' peptides represent the relative quantification of that protein.
[edit] Software
The data of the MS/MS spectra can be analyzed using software that is freely available
[edit] See also
[edit] References
- ^ Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004). "Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents". Mol. Cell. Proteomics 3 (12): 1154–69. doi:10.1074/mcp.M400129-MCP200. PMID 15385600.
- ^ Zieske LR (2006). "A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies". J. Exp. Bot. 57 (7): 1501–8. doi:10.1093/jxb/erj168. PMID 16574745.
- ^ Gafken PR, Lampe PD (2006). "Methodologies for characterizing phosphoproteins by mass spectrometry". Cell Commun. Adhes. 13 (5–6): 249–62. doi:10.1080/15419060601077917. PMC 2185548. PMID 17162667. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2185548.
- ^ Shadforth IP, Dunnley PJ, Lilley KS, Bessant C (2005). "i-Tracker: For quantitative proteomics using iTRAQ". BMC Genomics 6: 145. doi:10.1186/1471-2164-6-145. PMC 1276793. PMID 16242023. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1276793.
- ^ Muth, T., et al., jTraqX: a Free, Platform Independent Tool for Isobaric Tag Quantitation at the Protein Level, Proteomics, 2010, 10(6): 1223-1225, doi:10.1002/pmic.200900374
[edit] External links
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