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