Fig. 1.

Evaluation of phosphorylation-optimized MS²- and MS³-based TMT methods. a Colored peaks illustrate MSⁿ peak selection. MS² analysis either took place in the orbitrap (OT) or ion trap (IT). Ion selection for MS³ analysis was based on synchronous precursor selection (SPS) or neutral loss (NL)-triggered peak isolation. In the multiple charge state (MC) method, the MS³ isolation width was decreased for higher charge states. IT, OT and OT MC used multi-stage activation (MSA) with neutral loss mass 97.9673 Da. b Heatmap of correlation slopes of the 5% highest and lowest log2 ratios for all replicates. U2OS cells were treated 2 h with 5 µM doxorubicin (DOX) or DMSO (C). The resulting TMT sample was measured on an Orbitrap Fusion Lumos three times as technical replicates with each quantification method. c Bar plot showing the total number of quantified phosphopeptide DOX vs. C ratios per method for all replicates. d Violin plot showing log 10 signal-to-noise ratio distributions of the TMT reporter ions with the median marked as a dash