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Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.
Prabakaran S, Everley RA, Landrieu I, Wieruszeski JM, Lippens G, Steen H, Gunawardena J.
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The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites-the global pattern of phosphorylation or 'phospho-form'-giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2(n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods-western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)-on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.
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.
Baker,
Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.
2009, Pubmed
Baker,
Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.
2009,
Pubmed Bienkiewicz,
Random-coil chemical shifts of phosphorylated amino acids.
1999,
Pubmed Breuker,
Top-down identification and characterization of biomolecules by mass spectrometry.
2008,
Pubmed Cha,
Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.
2001,
Pubmed Cohen,
The regulation of protein function by multisite phosphorylation--a 25 year update.
2000,
Pubmed Cohen,
The role of protein phosphorylation in human health and disease. The Sir Hans Krebs Medal Lecture.
2001,
Pubmed Du,
Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function.
2005,
Pubmed Grangeasse,
Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology.
2007,
Pubmed Han,
Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons.
2006,
Pubmed Holmberg,
Multisite phosphorylation provides sophisticated regulation of transcription factors.
2002,
Pubmed Hunter,
The age of crosstalk: phosphorylation, ubiquitination, and beyond.
2007,
Pubmed Kim,
Phosphopeptide elution times in reversed-phase liquid chromatography.
2007,
Pubmed Kirkpatrick,
The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications.
2005,
Pubmed Landrieu,
NMR analysis of a Tau phosphorylation pattern.
2006,
Pubmed Lewis,
Signal transduction through MAP kinase cascades.
1998,
Pubmed Liokatis,
Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy.
2010,
Pubmed Mollica,
Are genuine changes in protein expression being overlooked? Reassessing Western blotting.
2009,
Pubmed Park,
Graded regulation of the Kv2.1 potassium channel by variable phosphorylation.
2006,
Pubmed Pesavento,
Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.
2008,
Pubmed Pesavento,
Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.
2006,
Pubmed Phanstiel,
Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.
2008,
Pubmed Pufall,
Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region.
2005,
Pubmed Selenko,
Looking into live cells with in-cell NMR spectroscopy.
2007,
Pubmed
,
Xenbase Selenko,
In situ observation of protein phosphorylation by high-resolution NMR spectroscopy.
2008,
Pubmed
,
Xenbase Shvartsburg,
Separation of peptide isomers with variant modified sites by high-resolution differential ion mobility spectrometry.
2010,
Pubmed Steen,
Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.
2005,
Pubmed Steen,
Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.
2006,
Pubmed Thomson,
Unlimited multistability in multisite phosphorylation systems.
2009,
Pubmed Timm,
Effect of high-accuracy precursor masses on phosphopeptide identification from MS3 spectra.
2010,
Pubmed Ulintz,
Comparison of MS(2)-only, MSA, and MS(2)/MS(3) methodologies for phosphopeptide identification.
2009,
Pubmed Van Regenmortel,
What is a B-cell epitope?
2009,
Pubmed Winter,
Separation of peptide isomers and conformers by ultra performance liquid chromatography.
2009,
Pubmed Wu,
Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways.
2004,
Pubmed Yao,
The ERK signaling cascade--views from different subcellular compartments.
2009,
Pubmed Young,
High throughput characterization of combinatorial histone codes.
2009,
Pubmed
