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Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

Nature Protocols volume 8, pages 461–480 (2013)Cite this article

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Abstract

Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere–based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO2). Here we present a detailed protocol for the preparation of monodisperse microsphere–based Ti4+-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ∼2 weeks to complete the protocol: 1 week to prepare the Ti4+-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.

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Figure 1: Depiction of the architecture of the Ti4+-IMAC adsorbent and the principle of phosphopeptide binding with Ti4+-IMAC.
Figure 2: The scheme of the preparation of Ti4+-IMAC adsorbents.
Figure 3: Characterization of the synthesized products by scanning electron microscopy.
Figure 4: The capacity of the assembled Ti4+-IMAC GELoader spin tip prepared as described in Steps 33 and 34 with 500 μg of packing material.
Figure 5: Flowchart briefly indicating the preparation and enrichment of a sample by Ti4+-IMAC.
Figure 6: The MALDI mass spectrometric analyses of a standard peptide mixture (consisting of varying amounts of α-casein and BSA digests) with and without enrichment.
Figure 7: LC-MS analysis of an unstimulated K562 cell lysate digest subjected to Ti4+-IMAC spin tips using a UHPLC system coupled to a Q-Exactive and differing gradient lengths.
Figure 8: Venn diagram analysis of analytical triplicate MS analyses using varying gradient lengths and the accumulative results of triplicate 60-min, 90-min, 120-min and 180-min gradients.
Figure 9: SCX/Ti4+-IMAC approach applied to a triple dimethyl-labeled MCF-7 sample.
Figure 10: Phosphorylation site localization.

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References

  1. Hunter, T. Signaling–2000 and beyond. Cell 100, 113–127 (2000).

    CAS  PubMed  Google Scholar 

  2. Pawson, T. & Scott, J.D. Protein phosphorylation in signaling–50 years and counting. Trends Biochem. Sci. 30, 286–290 (2005).

    CAS  PubMed  Google Scholar 

  3. Cohen, P. The regulation of protein function by multisite phosphorylation–a 25 year update. Trends Biochem. Sci. 25, 596–601 (2000).

    CAS  PubMed  Google Scholar 

  4. Lemeer, S. & Heck, A.J. The phosphoproteomics data explosion. Curr. Opin. Chem. Biol. 13, 414–420 (2009).

    CAS  PubMed  Google Scholar 

  5. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Reinders, J. & Sickmann, A. State-of-the-art in phosphoproteomics. Proteomics 5, 4052–4061 (2005).

    CAS  PubMed  Google Scholar 

  7. Temporini, C., Callerli, E., Massolini, G. & Caccialanza, G. Integrated analytical strategies for the study of phosphorylation and glycosylation in proteins. Mass Spectrom. Rev. 27, 207–236 (2008).

    CAS  PubMed  Google Scholar 

  8. Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101 (2005).

    CAS  PubMed  Google Scholar 

  9. Rikova, K. et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131, 1190–1203 (2007).

    CAS  PubMed  Google Scholar 

  10. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).

    CAS  PubMed  Google Scholar 

  11. Mohammed, S. & Heck, A. Jr. Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modifications. Curr. Opin. Biotechnol. 22, 9–16 (2011).

    CAS  PubMed  Google Scholar 

  12. Ballif, B.A., Villen, J., Beausoleil, S.A., Schwartz, D. & Gygi, S.P. Phosphoproteomic analysis of the developing mouse brain. Mol. Cell Proteomics 3, 1093–1101 (2004).

    CAS  PubMed  Google Scholar 

  13. Andersson, L. & Porath, J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 154, 250–254 (1986).

    CAS  PubMed  Google Scholar 

  14. Posewitz, M.C. & Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem. 71, 2883–2892 (1999).

    CAS  PubMed  Google Scholar 

  15. Dunn, J.D., Watson, J.T. & Bruening, M.L. Detection of phosphopeptides using Fe(III)-nitrilotriacetate complexes immobilized on a MALDI plate. Anal. Chem. 78, 1574–1580 (2006).

    CAS  PubMed  Google Scholar 

  16. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    CAS  PubMed  Google Scholar 

  17. Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B. & Heck, A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935–3943 (2004).

    CAS  PubMed  Google Scholar 

  18. Kweon, H.K. & Hakansson, K. Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 78, 1743–1749 (2006).

    CAS  PubMed  Google Scholar 

  19. Ficarro, S.B., Parikh, J.R., Blank, N.C. & Marto, J.A. Niobium(V) oxide (Nb2O5): application to phosphoproteomics. Anal. Chem. 80, 4606–4613 (2008).

    CAS  PubMed  Google Scholar 

  20. Mazanek, M. et al. Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis. Nat. Protoc. 2, 1059–1069 (2007).

    CAS  PubMed  Google Scholar 

  21. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886 (2005).

    CAS  PubMed  Google Scholar 

  22. Sugiyama, N. et al. Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol. Cell. Proteomics 6, 1103–1109 (2007).

    CAS  PubMed  Google Scholar 

  23. Thingholm, T.E., Jensen, O.N., Robinson, P.J. & Larsen, M.R. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol. Cell Proteomics 7, 661–671 (2008).

    CAS  PubMed  Google Scholar 

  24. Klemm, C. et al. Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides. J. Mass. Spectrom. 41, 1623–1632 (2006).

    CAS  PubMed  Google Scholar 

  25. Barnouin, K.N. et al. Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol. Proteomics 5, 4376–4388 (2005).

    CAS  PubMed  Google Scholar 

  26. Iliuk, A.B., Martin, V.A., Alicie, B.M., Geahlen, R.L. & Tao, W.A. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Mol. Cell. Proteomics 9, 2162–2172 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Wilson-Grady, J.T., Villen, J. & Gygi, S.P. Phosphoproteome analysis of fission yeast. J. Proteome Res. 7, 1088–1097 (2008).

    CAS  PubMed  Google Scholar 

  28. Gagnon, K.J., Perry, H.P. & Clearfield, A. Conventional and unconventional metal-organic frameworks based on phosphonate ligands: MOFs and UMOFs. Chem. Rev. 112, 1034–1054 (2012).

    CAS  PubMed  Google Scholar 

  29. Queffelec, C., Petit, M., Janvier, P., Knight, D.A. & Bujoli, B. Surface modification using phosphonic acids and esters. Chem. Rev. 112, 3777–3807 (2012).

    CAS  PubMed  Google Scholar 

  30. Nakayama, H. et al. Structural study of phosphate groups in layered metal phosphates by high-resolution solid-state P-31 NMR spectroscopy. J. Mater. Chem. 7, 1063–1066 (1997).

    CAS  Google Scholar 

  31. Clearfield, A. & Smith, G.D. The crystallography and structure of a-zirconium bis(monohydrogen orthophosphate) monohydrate. Inorg. Chem. 8, 431–436 (1969).

    CAS  Google Scholar 

  32. Nonglaton, G. et al. New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces. J. Am. Chem. Soc. 126, 1497–1502 (2004).

    CAS  PubMed  Google Scholar 

  33. Cinier, M. et al. Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays. Bioconjugate Chem. 20, 2270–2277 (2009).

    CAS  Google Scholar 

  34. Wang, Q.F., Zhong, L., Sun, J.Q. & Shen, J.C. A facile layer-by-layer adsorption and reaction method to the preparation of titanium phosphate ultrathin films. Chem. Mater. 17, 3563–3569 (2005).

    CAS  Google Scholar 

  35. Zhou, H. et al. Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis. J. Proteome Res. 5, 2431–2437 (2006).

    CAS  PubMed  Google Scholar 

  36. Feng, S. et al. Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis. Mol. Cell Proteomics 6, 1656–1665 (2007).

    CAS  PubMed  Google Scholar 

  37. Zhao, L. et al. The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis. J. Am. Soc. Mass. Spectrom. 19, 1176–1186 (2008).

    CAS  PubMed  Google Scholar 

  38. Dong, J., Zhou, H., Wu, R., Ye, M. & Zou, H. Specific capture of phosphopeptides by Zr4+-modified monolithic capillary column. J. Sep. Sci. 30, 2917–2923 (2007).

    CAS  PubMed  Google Scholar 

  39. Zhou, H. et al. Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis. J. Proteome Res. 7, 3957–3967 (2008).

    CAS  PubMed  Google Scholar 

  40. Yu, Z. et al. Preparation of monodisperse immobilized Ti(4+) affinity chromatography microspheres for specific enrichment of phosphopeptides. Anal. Chim. Acta 636, 34–41 (2009).

    CAS  PubMed  Google Scholar 

  41. Zhou, H. et al. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. Mol. Cell Proteomics 10, M110 006452 (2011).

    PubMed  PubMed Central  Google Scholar 

  42. Wang, Q.C., Hosoya, K., Svec, F. & Frechet, J.M. Polymeric porogens used in the preparation of novel monodispersed macroporous polymeric separation media for high-performance liquid chromatography. Anal. Chem. 64, 1232–1238 (1992).

    CAS  PubMed  Google Scholar 

  43. Ugelstad, J., Söderberg, L., Berge, A. & Bergström, J. Monodisperse polymer particles-A step forward for chromatography. Nature 24, 95–96 (1983).

    Google Scholar 

  44. Phanstiel, D.H. et al. Proteomic and phosphoproteomic comparison of human ES and iPS cells. Nat. Methods 8, 821–827 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Yu, Y. et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 332, 1322–1326 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Benschop, J.J. et al. Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis. Mol. Cell. Proteomics 6, 1198–1214 (2007).

    CAS  PubMed  Google Scholar 

  47. Olsen, J.V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    CAS  PubMed  Google Scholar 

  48. Huttlin, E.L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Dephoure, N. et al. A quantitative atlas of mitotic phosphorylation. Proc. Natl. Acad. Sci. USA 105, 10762–10767 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Grosstessner-Hain, K. et al. Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome. Mol. Cell Proteomics 10, M111 008540 (2011).

    PubMed  PubMed Central  Google Scholar 

  51. Lundby, A. et al. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. Nat. Commun. 3, 876 (2012).

    PubMed  Google Scholar 

  52. Ugelstad, J., Mórk, P.C., Herder Kaggerud, K., Ellingsen, T. & Berge, A. Swelling of oligomer-polymer particles. New methods of preparation. Adv. Colloid Interface Sci. 13, 101–140 (1980).

    CAS  Google Scholar 

  53. Han, G. et al. Comprehensive and reliable phosphorylation site mapping of individual phosphoproteins by combination of multiple stage mass spectrometric analysis with a target-decoy database search. Anal. Chem. 81, 5794–5805 (2009).

    CAS  PubMed  Google Scholar 

  54. Gauci, S. et al. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal. Chem. 81, 4493–4501 (2009).

    CAS  PubMed  Google Scholar 

  55. Molina, H., Horn, D.M., Tang, N., Mathivanan, S. & Pandey, A. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2199–2204 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Song, C. et al. Reversed-phase-reversed-phase liquid chromatography approach with high orthogonality for multidimensional separation of phosphopeptides. Anal. Chem. 82, 53–56 (2010).

    CAS  PubMed  Google Scholar 

  57. McNulty, D.E. & Annan, R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol. Cell. Proteomics 7, 971–980 (2008).

    CAS  PubMed  Google Scholar 

  58. Villen, J. & Gygi, S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    PubMed  PubMed Central  Google Scholar 

  59. Hennrich, M.L., van den Toorn, H.W.P., Groenewold, V., Heck, A.J.R. & Mohammed, S. Ultra acidic strong cation exchange enabling the efficient enrichment of basic phosphopeptides. Anal. Chem. 84, 1804–1808 (2012).

    CAS  PubMed  Google Scholar 

  60. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1, 376–386 (2002).

    CAS  PubMed  Google Scholar 

  61. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    CAS  PubMed  Google Scholar 

  62. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics 3, 1154–1169 (2004).

    CAS  PubMed  Google Scholar 

  63. Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).

    CAS  PubMed  Google Scholar 

  64. Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).

    CAS  PubMed  Google Scholar 

  65. Boersema, P.J., Mohammed, S. & Heck, A.J. Phosphopeptide fragmentation and analysis by mass spectrometry. J. Mass Spectrom. 44, 861–878 (2009).

    CAS  PubMed  Google Scholar 

  66. Swaney, D.L., Wenger, C.D., Thomson, J.A. & Coon, J.J. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 106, 995–1000 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Chi, A. et al. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2193–2198 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Nagaraj, N., D′Souza, R.C., Cox, J., Olsen, J.V. & Mann, M. Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. J. Proteome Res. 9, 6786–6794 (2010).

    CAS  PubMed  Google Scholar 

  69. Jedrychowski, M.P. et al. Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics. Mol. Cell Proteomics 10, M111 009910 (2011).

    PubMed  PubMed Central  Google Scholar 

  70. Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J. & Gygi, S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).

    CAS  PubMed  Google Scholar 

  71. Savitski, M.M. et al. Confident phosphorylation site localization using the Mascot Delta Score. Mol. Cell Proteomics 10, M110 003830 (2011).

    PubMed  Google Scholar 

  72. Taus, T. et al. Universal and confident phosphorylation site localization using phosphoRS. J. Proteome Res. 10, 5354–5362 (2011).

    CAS  PubMed  Google Scholar 

  73. Raijmakers, R. et al. Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity. Mol. Cell. Proteomics 7, 1755–1762 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  PubMed  Google Scholar 

  75. Kocher, T., Pichler, P., Swart, R. & Mechtler, K. Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients. Nat. Protoc. 7, 882–890 (2012).

    PubMed  Google Scholar 

  76. Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    CAS  PubMed  Google Scholar 

  77. Eng, J.K., McCormack, A.L. & Yates, J.R. An approach to correlate MS/MS data to amino acid sequences in proten database. J. Am. Soc. Mass. Spectrom. 5, 976–989 (1994).

    CAS  PubMed  Google Scholar 

  78. Craig, R. & Beavis, R.C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).

    CAS  PubMed  Google Scholar 

  79. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  PubMed  Google Scholar 

  80. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    CAS  PubMed  Google Scholar 

  81. Kall, L., Canterbury, J.D., Weston, J., Noble, W.S. & MacCoss, M.J. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat. Methods 4, 923–925 (2007).

    PubMed  Google Scholar 

  82. Song, C. et al. Systematic analysis of protein phosphorylation networks from phosphoproteomic data. Mol. Cell Proteomics 11, 1070–1083 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Kettenbach, A.N. & Gerber, S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal. Chem. 83, 7635–7644 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Kjellstrom, S. & Jensen, O.N. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins. Anal. Chem. 76, 5109–5117 (2004).

    PubMed  Google Scholar 

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Acknowledgements

This work was supported in part by the PRIME-XS project with the grant agreement number 262067, funded by the European Union 7th Framework Program; The Netherlands Proteomics Centre, embedded in the Netherlands Genomics Initiative; the Netherlands Organization for Scientific Research (NWO) with the VIDI grant (700.10.429); the Creative Research Group Project by the National Natural Sciences Foundation of China (21021004); and a China State Key Basic Research Program grant (2012CB910101, 2013CB911202).

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  1. Houjiang Zhou

    Present address: Present address: Department of Biochemistry, University of Cambridge, Cambridge, UK.,

  2. Houjiang Zhou and Mingliang Ye: These authors contributed equally to this work.

Authors and Affiliations

  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Houjiang Zhou, Eleonora Corradini, Alba Cristobal, Albert J R Heck & Shabaz Mohammed

  2. Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

    Houjiang Zhou, Mingliang Ye, Jing Dong & Hanfa Zou

  3. Netherlands Proteomics Centre, Utrecht, The Netherlands

    Houjiang Zhou, Eleonora Corradini, Alba Cristobal, Albert J R Heck & Shabaz Mohammed

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Contributions

H. Zhou, M.Y., S.M. and H. Zou designed the studies. H. Zhou performed the phosphoproteomic experiments and analyzed the data. J.D. carried out the synthesis experiment. E.C. and A.C. assisted in the Q-Exactive experiments. All authors discussed experimental results. A.J.R.H., H. Zou and S.M. supervised the project and wrote the manuscript with H. Zhou and M.Y.

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Zhou, H., Ye, M., Dong, J. et al. Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography. Nat Protoc 8, 461–480 (2013). https://doi.org/10.1038/nprot.2013.010

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