Proteomic Analysis of Posttranslational Modifications Using iTRAQ in Leishmania
iTRAQ is a high coverage quantitative proteomics technique identifies and quantitates abundance changes of multiple (up to eight) distinct protein samples. To date, one iTRAQ-MS/MS assay can identify up to quarter of cells proteome. Each of the eight tags covalently binds to the N-terminus as well as arginine and lysine side chains of peptides, enabling labeling of the entire peptide population in each sample. Following tagging, the various protein samples are mixed and subjected to LC-MS/MS analysis. In the first round identical peptides from the different protein populations focus in a single pick. Subsequently, sequence of each peptide is determined. The tags whose m/z is similar to that of natural amino acids are used to determine relative abundance. To date, iTRAQ enabled identification of almost 2,000 Leishmania proteins. Here, we provide protocols for protein abundance changes and for phosphoproteomics analysis in Leishmania parasites.
Key wordsLeishmania iTRAQ Affinity tag Proteomics Phosphoproteomics Protein expression Quantitative proteomics
I thank Dr. Polina Tsigankov for critical reading. I thanks Drs. Christoph ** and *** for providing me their protocols. This work was supported by U.S.-Israel Binational Foundation grant 2009226.
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