Figure 6.

NMR analysis and four-site phospho-form distribution. (A) Phospho-form distributions of each of the four Erk samples obtained by NMR. The pTpY phospho-form was determined from three resonances, as tabulated below with their normalised peak integral values. The additional A and T resonances could not be fully confirmed in the Erk-TY and Erk-TpY samples without 3D spectra and were therefore not measured, as indicated by a dash. The pTY phospho-form was determined by the single resonance pT(EY), as also tabulated. The TY and TpY phospho-forms were not detectable, indicated by the asterisks in the distributions. The bars show either a single data point or the mean±s.d. Measurements agree to within 10% with those found by pepMS (Figure 2B). (B) The four-site phospho-form distribution of Erk-pTpY. The 16 phospho-forms are listed horizontally, in the form (p)S(p)S(p)T(p)Y, with their corresponding proportions denoted by a₁,L,a₁₆. These values were calculated by combining information from pepMS and proMS for both Erk-pTpY and Erk-pTY, as explained in Supplementary Method 1.2. Grey columns indicate phospho-forms whose individual proportions can be determined or placed between upper and lower bounds. The dashed columns and the columns with a dotted outline indicate two sets of phospho-forms whose individual values could not be determined, but whose sums were limited as shown in the equations. The asterisks mark the phospho-forms on which most of the distribution is concentrated, whose relevance is discussed in the text.