Figure 2.

Peptide-based MS analysis of Erk. (A) Equal amounts of each of the synthetic internal standards were subjected to LC/MS. The left panel shows three superimposed extracted ion chromatograms for the m/z values expected for 0 (red), 1 (blue) and 2 (green) phosphorylations. Each individual chromatogram has been normalised to the intensity of its maximal peak. The singly phosphorylated peptides were identified by MS². The right panels show the normalised MS² spectra for the z=3 charge state (m/z=745.33) eluting around 24.2 min (top) and around 20.9 min (bottom). The top spectrum shows the neutral loss of phosphoric acid (m/z=712.7) that characterises pTY. Its absence in the bottom spectrum characterises TpY. (B) Phospho-form distributions of each of the four Erk samples. The peak area of the phospho-form was divided by the peak area of the corresponding internal standard, and the four ratios for each phospho-form were then sum normalised as proportions of the total. Data were pooled from experiments carried out on different days, with five replicates for Erk-TY and Erk-pTpY and three replicates for Erk-pTY and Erk-TpY; the error bars give the mean±s.d. of the corresponding normalised values. (C) The values in B have been rearranged for comparison with Figure 1D. Each vertical sub-panel shows the results for the phospho-form listed at the top of the sub-panel, for each of the four samples, as listed horizontally. The arrows show the corresponding comparisons with those in Figure 1D. Source data is available for this figure at www.nature.com/msb.

Source data for Figure 2B ^{(17.5KB, xls)}