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 ((15)N, (13)C) 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 (15)N- and (13)C-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, (12)C(8)H(16)(15)N(1)(+); TMT127H, (12)C(7)(13)C(1)H(16)(14)N(1)(+); TMT129L, (12)C(6)(13)C(2)H(16)(15)N(1)(+); and TMT129H, (12)C(5)(13)C(3)H(16)(14)N(1)(+)). 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.