Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/iTRAQ

iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel based technique used to identify and quantify proteins from different sources in one single experiment. It uses isotope coded covalent tags. iTRAQ is used in proteomics to study quantitative changes in the proteome.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 Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol. Cell. Proteomics 3 12 1154–69 2004 15385600 10.1074/mcp.M400129-MCP200Zieske LR A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies J. Exp. Bot. 57 7 1501–8 2006 16574745 10.1093/jxb/erj168Gafken PR, Lampe PD Methodologies for characterizing phosphoproteins by mass spectrometry Cell Commun. Adhes. 13 5-6 249–62 2006 17162667 10.1080/15419060601077917 2185548 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, using software such as the freely available i-TrackerShadforth IP, Dunnley PJ, Lilley KS, Bessant C i-Tracker: For quantitative proteomics using iTRAQ BMC Genomics 6 145 2005 10.1186/1471-2164-6-145 16242023 1276793. References