Isobaric tagging

The protocol for using isobaric tags differs from that described previously for the ICAT or ECAT type reagents. In the following method, the proteins are denatured and the disulfides reduced and then alkylated to block them permanently. This eliminates disulfide re-association and also prevents the isobaric tags from forming thioester modification with cysteine thiols. Next, the proteins are digested with trypsin and then modified with an isobaric tag. Each sample is labeled with a different isobaric compound so that the samples can be differentiated upon MS/MS analysis. 

Dissolve the isobaric tagging reagents in acetonitrile or ethanol at a concentration of 50 mM (or according to the manufacturer s recommendations). Use a fume hood to handle organic solvents. 

Add a quantity of the appropriate isobaric tag solution to each sample to provide a final concentration of 10-20 mM. This quantity of reagent will assure a large molar excess of reagent over the concentration of peptides present in order to modify completely all peptides at their N-terminus. Note that the e-amino groups of lysine residues also will be modified by this procedure. React for 1 hour at room temperature. 

Ross, P.L., Huang, Y.N., Marchese, J.N., 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., and Pappin, D.J. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154-1169. 

Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharmyces cerevisiae using amino-reactive isobaric tagging reagents. Mol Cell Proteomics 3.12 1154-1169... 

Isobaric tags for relative and absolute quantitation of proteins in proteomic research Janus kinase c-Jun iV-terminal kinase... 

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