High-resolution enabled TMT 8-plexing.

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (N-15, C-13) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between N-15- and C-13-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, (C8H16N1+)-C-12-N-15; TMT127H, (C7C1H16N1+)-C-12-C-13-N-14; TMT129L, (C6C2H16N1+)-C-12-C-13-N-15; and TMT129H, (C5C3H16N1+)-C-12-C-13-N-14). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays.