Prabakaran S , Everley RA , Landrieu I , Wieruszeski JM , Lippens G , Steen H , Gunawardena J .

R01 GM081578-04 NIGMS NIH HHS , R01-GM081578 NIGMS NIH HHS , T32 HL076115 NHLBI NIH HHS , R01 GM094844 NIGMS NIH HHS , R01 GM081578 NIGMS NIH HHS

	Figure 1. Antibody analysis of Erk. (A) Primary sequence of the His6-tagged Erk2 from Xenopus laevis used in the paper, showing two tryptic peptides identified as being phosphorylated (underlined): the central peptide contains the two activating phosphorylation sites in the canonical TEY motif and the N-terminal peptide (omitting the N-terminal M, which is cleaved) contains the His6 tag and the two novel S-phosphorylation sites discussed in the text. The phospho-sites are marked in large, bold, blue font. (B) Ribbon diagram of Erk2 from Rattus norvegicus (PDB-2ERK) showing activating phosphorylations on T and Y (yellow). (C) Summary of amount/intensity relationship for each antibody. The measures R2 and α are explained in the text and derived from the data in Supplementary Figure 1. (D) Phospho-form measurements. Each vertical sub-panel shows the results from the antibody listed at the top of the sub-panel. Erk samples are listed horizontally at the bottom with X denoting sample Erk-X. Each bar gives the ratio of the fluorescence intensity of the corresponding antibody to the intensity of the anti-total Erk antibody. Both antibodies were multiplexed on the same gel. Each of the four Erk samples were run in triplicate on the same gel and each gel was repeated three times; error bars give the mean±s.d. of the nine data points. The underlying data are shown in Supplementary Figure 2. Note that there is no sum normalisation and it is coincidental that values lie between 0 and 1. The arrows show particular comparisons discussed in the text. Source data is available for this figure at www.nature.com/msb.Source data for Figure 1D.
	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.
	Figure 3. Comparison of pepMS and antibodies. (A) Rearrangement of the phospho-form distributions (Supplementary Figure 3) of the second, non-isotopically labelled sample set. For each phospho-form listed at the top of each sub-panel, the normalised values of the phospho-form for each sample are collected together. Samples are listed at the bottom of the figure. (B) Antibody data for the second sample set, as in Figure 1D. (C) Antibody data for the second sample set, after spiking with whole-cell lysate. The vertical scales for B and C lie between 0 and 1 only by coincidence; there is no sum-normalisation, as there is for A. The arrows show comparisons discussed in the text. Similar results were obtained using HRP-conjugated antibodies and CCD imaging (Supplementary Figure 4). Source data is available for this figure at www.nature.com/msb.Source data for Figure 3B. Source data for Figure 3C.
	Figure 4. Protein-based MS analysis of Erk. (A) Mass/charge spectrum of intact Erk-pTpY, showing multiple charge states. (B) Neutral mass spectrum of Erk-pTpY, after charge-state deconvolution. The calculated masses of 15N-labelled His6-tagged Erk2, with 0 to 4 phosphorylations are listed in the inset. Those for 2P, 3P and 4P agree to within 100 p.p.m. with the peaks marked in the spectrum. The positions that would have been occupied by the 0P and 1P states are shown by vertical arrows. Three of the remaining peaks correspond to oxidation products (+32Da, marked by asterisks). (C) Sum-normalised distributions of isobaric groups for each of the four samples, showing 0–4 phosphorylations. Only two phosphorylation sites were detected in the Erk-TY and Erk-TpY samples and the insets compare the proMS values with those calculated from the pepMS data in Figure 2B. Five replicates were used for Erk-TY and Erk-pTpY and the error bars show mean±s.d. Due to insufficient material, only two replicates were possible for Erk-pTY and Erk-TpY. The corresponding columns show the mean, with the two data points listed above to show the variation. Source data is available for this figure at www.nature.com/msb.Source data for Figure 4C.
	Figure 5. NMR spectra of Erk. (A) Superimposed 1H/15N HSQC spectra of Erk-pTpY (black) and Erk-TY (red). Blue circles and arrows mark the C-terminal Y resonance used for normalisation (Supplementary Figure 8) and the additional A and T resonances used for quantifying pTpY (Supplementary Methods 1.1). The small box in the centre shows a major and a minor peak that are present in Erk-pTpY but not in Erk-TY, in the region above 8.8 δ 1H, where pS and pT are found. The box is enlarged in inset 2. Inset 1 shows a section of the 3D HNCACB spectrum, with the peaks corresponding to the Cα and Cβ of the major peak in Erk-pTpY (vertical black dashed line). This identifies the major peak as coming from pT. Inset 3 shows the region corresponding to inset 2 from the 1H/15N HSQC spectrum of Erk-pTY. Supplementary Figure 6 explains why the major peak of Erk-pTY and the minor peak of Erk-pTpY, linked by the vertical blue dashed line, represent pT(EY), while the major peak in the Erk-pTpY sample represents pT(EpY). Additional peaks below the small box do not titrate with pH and therefore do not correspond to phosphorylated residues. The horizontal scales on all three insets are the same (δ 1H in p.p.m.), but the vertical scale on inset 1 is δ 13C in p.p.m., while on insets 2 and 3 it is δ 15N in p.p.m. (B) Superimposition of three 1H/15N HSQC spectra, colour coded as described in the legend. Note that the horizontal and vertical ranges are different from the insets in part a. The minor peak in the Erk-pTpY spectrum falls on the same pH titration line as the major peaks in the Erk-pTY spectra, supporting the assignment in A. The resonances in the two pH titration lines at the top (arrows) show that there are at least two more pS or pT residues in addition to that on the canonical TEY motif, as discussed in the text. These additional peaks could not be assigned by NMR as their 3D HNCACB intensities were too low.
	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.

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