Fig. 2.

Benchmarking Tomahto by measuring protein levels for 77 kinases across three human cell lines. (A) Lysates from biological triplicates of HCT116 and MCF7 and quadruplicates of HEK293T were processed and labeled with TMT10 reagents. The Tomahto assay was performed with 154 spiked-in trigger peptides corresponding to 77 kinases and a 2-h single-shot method. In addition, shotgun analysis was performed on the same samples after fractionation. Twelve fractions were analyzed with 3-h gradients using the standard DDA-SPS-MS3 method (36 h total). (B) Representative trigger and target MS2s. These are used to confirm the identity of both the trigger and the target precursor peaks. The target MS2 is often of very low abundance and requires long injections times (812 ms here). Note that all labeled fragment masses differ by 6 Da between plots due to the TMTsh labeling on both termini. (C) Overlap for the kinases quantified by both methods. (D) Correlation between standard DDA-SPS-MS3 (fractionated, 36-h analysis) and Tomahto (unfractionated, 2-h analysis). (E) Hierarchical clustering of kinases quantified by Tomahto. Replicates perfectly clustered and many signature up- or down-regulated kinases were identified. (F) Bar plots of example proteins, EGFR, MAPK3, SRC, and EPHA2.