Supporting Information

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Fig. 7. Example of quantification methodology. By using XCALIBUR software, monoisotopic peaks (from the Fourier transform-ion cyclotron resonance MS scan) of the unlabeled (blue circle) and labeled (red triangle) samples were identified for a specific phosphopeptide for each time point. An average scan was formed to include each scan in an individual run where the phosphopeptide could be unambiguously identified. Both the unlabeled and labeled monoisotopic peaks are integrated in XCALIBUR, and the area is displayed (see integrated peaks and area values at right). Starting with the unstimulated samples, dividing the area of the unlabeled monoisotopic peak by the area of the labeled monoisotopic peak gives a value of 1.57. This ratio is set to 100%. The ratio at 1 min after EGF stimulation is 2.89. Division of this number by 1.57 gives 1.84 or 184% relative to the unstimulated ratio. This calculation is performed for each time point. It is important to note that many activating phosphorylation events may not be detectable in a fully quiescent state. In such cases, one must make conservative estimates for ratios based on calculations of signal to noise.

Fig. 8. Tandem MS spectrum of a doubly charged RSK1 peptide ion showing a previously uncharacterized phosphorylation event at Ser-369.

Fig. 9. Tandem MS spectrum of a triply charged tuberous sclerosis complex 2 (TSC2) peptide ion showing a previouly uncharacterized phosphorylation event at Ser-1364. Note peptide contains one [¹³C₆,¹⁵N₂]Lys and one [¹³C₆]Arg.

Fig. 10. Phosphorylation Profiling of tuberous sclerosis complex (TSC)1. Quantification of eight identified phosphopeptides in TSC1. The tandem MS spectrum for phosphopeptide "H" could not distinguish between phosphorylation at Thr-393 or Ser-394. We have thus placed parentheses in the sequence around those residues to indicate that the phosphorylation site could be one or the other. Phosphorylation at Ser-584 and Thr-1047 have been reported to be phosphorylated by cyclin-dependent kinase 1 (CDK1) in vivo. The other six phosphorylation sites have not been reported.