Cardiac Phosphoproteomics during Remote Ischemic Preconditioning: A Role for the Sarcomeric Z-Disk Proteins
Figure 1
Phospho-TMT workflow. (a) Structure of the TMT Tag. Six tags are available, each with a different reporter group (m/z 126, 127, 128, 129, 130, or 131). The mass normaliser region balances out the difference in mass in the reporter groups such that the overall tag mass is constant. The reactive group provides amine-specific labelling. (b) Six samples are digested with trypsin to generate peptides which are then labelled with one of the six TMT tags. The labelling reaction is quenched and the samples are pooled. The pooled sample is passed through a titanium dioxide (TiO2) column, phosphopeptides bind to the column while nonphosphorylated peptides pass straight through. The phosphopeptides are then eluted and analysed by LC-MS/MS. Differentially tagged peptides are indistinguishable at the MS1 level since the overall tag mass is constant. Fragmentation of the peptides detected in the MS1 spectra produces secondary MSMS spectra for each peptide, allowing elucidation of the peptide sequence. In addition, the fragmentation process causes cleavage of the linker region within the tag, releasing the reporter groups which appear as a cluster of ions at the low mass end of each MSMS spectra. The relative intensity of the members of this ion cluster shows the relative abundance of that peptide between the six samples under comparison.
