Enrichment Strategies in Phosphoproteomics

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1355)

Abstract

The comprehensive study of the phosphoproteome is heavily dependent on appropriate enrichment strategies that are most often, but not exclusively, carried out on the peptide level. In this chapter, I give an overview of the most widely used techniques. In addition to dedicated antibodies, phosphopeptides are enriched by their selective interaction with metals in the form of chelated metal ions or metal oxides. The negative charge of the phosphate group is also exploited in a variety of chromatographic fractionation methods that include different types of ion exchange chromatography, hydrophilic interaction chromatography (HILIC), and electrostatic repulsion HILIC (ERLIC) chromatography. Selected examples from the literature will demonstrate how a combination of these techniques with current high-performance mass spectrometry enables the identification of thousands of phosphorylation sites from various sample types.

Key words

Phosphopeptideenrichment Fractionation Sample preparation Mass spectrometry 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Kim M-S, Pinto SM, Getnet D et al (2014) A draft map of the human proteome. Nature 509:575–581PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509:582–587CrossRefPubMedGoogle Scholar
  3. 3.
    Beltran L, Cutillas PR (2012) Advances in phosphopeptide enrichment techniques for phosphoproteomics. Amino Acids 43:1009–1024CrossRefPubMedGoogle Scholar
  4. 4.
    Fila J, Honys D (2012) Enrichment techniques employed in phosphoproteomics. Amino Acids 43:1025–1047PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Engholm-Keller K, Larsen MR (2013) Technologies and challenges in large-scale phosphoproteomics. Proteomics 13:910–931CrossRefPubMedGoogle Scholar
  6. 6.
    Loroch S, Dickhut C, Zahedi RP, Sickmann A (2013) Phosphoproteomics—More than meets the eye. Electrophoresis 34:1483–1492CrossRefPubMedGoogle Scholar
  7. 7.
    Guo M, Huang BX (2013) Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation. Proteomics 13:424–437CrossRefPubMedGoogle Scholar
  8. 8.
    Roux PP, Thibault P (2013) The coming of age of phosphoproteomics—from large data sets to inference of protein functions. Mol Cell Proteomics 12:3453–3464PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Jünger MA, Aebersold R (2014) Mass spectrometry-driven phosphoproteomics: patterning the systems biology mosaic. Wiley Interdiscip Rev Dev Biol 3:83–112CrossRefPubMedGoogle Scholar
  10. 10.
    Wells JM, McLuckey SA (2005) Collision-induced dissociation (CID) of peptides and proteins. Methods Enzymol 402:148–185CrossRefPubMedGoogle Scholar
  11. 11.
    Boersema PJ, Mohammed S, Heck AJR (2009) Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom 44:861–878CrossRefPubMedGoogle Scholar
  12. 12.
    Chalkley RJ, Clauser KR (2012) Modification site localization scoring: strategies and performance. Mol Cell Proteomics 11:3–14PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Rush J, Moritz A, Lee KA et al (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23:94–101CrossRefPubMedGoogle Scholar
  14. 14.
    Boersema PJ, Foong LY, Ding VMY et al (2010) In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling. Mol Cell Proteomics 9:84–99PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Kee J-M, Oslund RC, Perlman DH et al (2013) A pan-specific antibody for direct detection of protein histidine phosphorylation. Nat Chem Biol 9:416–421PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Porath J, Carlsson J, Olsson I et al (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598–599CrossRefPubMedGoogle Scholar
  17. 17.
    Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254CrossRefPubMedGoogle Scholar
  18. 18.
    Zhou H, Ye M, Dong J et al (2008) Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis. J Proteome Res 7:3957–3967CrossRefPubMedGoogle Scholar
  19. 19.
    Zhou H, Ye M, Dong J et al (2013) Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography. Nat Protoc 8:461–480CrossRefPubMedGoogle Scholar
  20. 20.
    Iliuk AB, Martin VA, Alicie BM et al (2010) In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Mol Cell Proteomics 9:2162–2172PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Jayasundera KB, Iliuk AB, Nguyen A et al (2014) Global phosphoproteomics of activated b cells using complementary metal ion functionalized soluble nanopolymers. Anal Chem 86:6363–6371PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Tsai C-F, Hsu C-C, Hung J-N et al (2014) Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography. Anal Chem 86:685–693CrossRefPubMedGoogle Scholar
  23. 23.
    Pinkse MWH, Uitto PM, Hilhorst MJ et al (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943CrossRefPubMedGoogle Scholar
  24. 24.
    Larsen MR, Thingholm TE, Jensen ON et al (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886CrossRefPubMedGoogle Scholar
  25. 25.
    Leitner A (2010) Phosphopeptide enrichment using metal oxide affinity chromatography. Trends Anal Chem 29:177–185CrossRefGoogle Scholar
  26. 26.
    Sugiyama N, Masuda T, Shinoda K et al (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6:1103–1109CrossRefPubMedGoogle Scholar
  27. 27.
    Fukuda I, Hirabayashi-Ishioka Y, Sakikawa I et al (2013) Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis. J Proteome Res 12:5587–5597CrossRefPubMedGoogle Scholar
  28. 28.
    Kyono Y, Sugiyama N, Imami K et al (2008) Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography. J Proteome Res 7:4585–4593CrossRefPubMedGoogle Scholar
  29. 29.
    Imami K, Sugiyama N, Kyono Y et al (2008) Automated phosphoproteome analysis for cultured cancer cells by two-dimensional NanoLC-MS using a calcined titania/C18 Biphasic column. Anal Sci 24:161–166CrossRefPubMedGoogle Scholar
  30. 30.
    Li Q-R, Ning Z-B, Tang J-S et al (2009) Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity. J Proteome Res 8:5375–5381CrossRefPubMedGoogle Scholar
  31. 31.
    Schmidt A, Trentini DB, Spiess S et al (2014) Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response. Mol Cell Proteomics 13:537–550PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101:12130–12135PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Olsen JV, Blagoev B, Gnad F et al (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648CrossRefPubMedGoogle Scholar
  34. 34.
    Han G, Ye M, Zhou H et al (2008) Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography. Proteomics 8:1346–1361CrossRefPubMedGoogle Scholar
  35. 35.
    Boersema PJ, Mohammed S, Heck AJR (2008) Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Anal Bioanal Chem 391:151–159PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    McNulty DE, Annan RS (2008) Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics 7:971–980CrossRefPubMedGoogle Scholar
  37. 37.
    Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80:62–76CrossRefPubMedGoogle Scholar
  38. 38.
    Zhang X, Ye J, Jensen ON et al (2007) Highly efficient phosphopeptide enrichment by calcium phosphate precipitation combined with subsequent IMAC enrichment. Mol Cell Proteomics 6:2032–2042CrossRefPubMedGoogle Scholar
  39. 39.
    Ruse CI, McClatchy DB, Lu B et al (2008) Motif-specific sampling of phosphoproteomes. J Proteome Res 7:2140–2150PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Güzel Y, Rainer M, Mirza MR et al (2013) Highly selective recovery of phosphopeptides using trypsin-assisted digestion of precipitated lanthanide-phosphoprotein complexes. Analyst 138:2897–2905CrossRefPubMedGoogle Scholar
  41. 41.
    Leitner A, Lindner W (2009) Chemical tagging strategies for mass spectrometry-based phospho-proteomics. Methods Mol Biol 527:229–243CrossRefPubMedGoogle Scholar
  42. 42.
    Zhou H, Watts JD, Aebersold R (2001) A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19:375–378CrossRefPubMedGoogle Scholar
  43. 43.
    Tao WA, Wollscheid B, O‘Brien R et al (2005) Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat Methods 2:591–598CrossRefPubMedGoogle Scholar
  44. 44.
    Nika H, Lee J, Willis IM et al (2012) Phosphopeptide characterization by mass spectrometry using reversed-phase supports for solid-phase β-elimination/michael addition. J Biomol Tech 23:51–68PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Nika H, Nieves E, Hawke DH et al (2013) Optimization of the β-elimination/michael addition chemistry on reversed-phase supports for mass spectrometry analysis of O-linked protein modifications. J Biomol Tech 24:132–153PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Fonslow BR, Niessen SM, Singh M et al (2012) Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT. J Proteome Res 11:2697–2709PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Chen Q, Wei C, Zhang Y et al (2014) Single-crystalline hyperbranched nanostructure of iron hydroxyl phosphate Fe5(PO4)4(OH)3 x 2 H2O for highly selective capture of phosphopeptides. Sci Rep 4:3753PubMedCentralPubMedGoogle Scholar
  48. 48.
    Li X-S, Xu L-D, Zhu G-T et al (2012) Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides. Analyst 137:959–967CrossRefPubMedGoogle Scholar
  49. 49.
    Li L-P, Zheng T, Xu L-N et al (2013) SnO2-ZnSn(OH)6: a novel binary affinity probe for global phosphopeptide detection. Chem Commun 49:1762–1764CrossRefGoogle Scholar
  50. 50.
    Cheng G, Li S-M, Wang Y et al (2013) REPO4 (RE = La, Nd, Eu) affinity nanorods modified on a MALDI plate for rapid capture of target peptides from complex biosamples. Chem Commun 49:8492–8494CrossRefGoogle Scholar
  51. 51.
    Cheng G, Liu Y-L, Wang Z-G et al (2013) Yolk-shell magnetic microspheres with mesoporous yttrium phosphate shells for selective capture and identification of phosphopeptides. J Mater Chem B 1:3661–3669CrossRefGoogle Scholar
  52. 52.
    Fischnaller M, Köck R, Bakry R et al (2014) Enrichment and desalting of tryptic protein digests and the protein depletion using boron nitride. Anal Chim Acta 823:40–50CrossRefPubMedGoogle Scholar
  53. 53.
    Furuhashi T, Nukarinen E, Ota S et al (2014) Boron nitride as desalting material in combination with phosphopeptide enrichment in shotgun proteomics. Anal Biochem 452:16–18CrossRefPubMedGoogle Scholar
  54. 54.
    Li Q-R, Ning Z-B, Yang X-L (2012) Complementary workflow for global phosphoproteome analysis. Electrophoresis 33:3291–3298CrossRefPubMedGoogle Scholar
  55. 55.
    Thingholm TE, Jensen ON, Robinson PJ et al (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics 7:661–671CrossRefPubMedGoogle Scholar
  56. 56.
    Engholm-Keller K, Birck P, Storling J et al (2012) TiSH—a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J Proteomics 75:5749–5761CrossRefPubMedGoogle Scholar
  57. 57.
    Mertins P, Qiao JW, Patel J et al (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10:634–637PubMedCentralCrossRefPubMedGoogle Scholar
  58. 58.
    Swaney DL, Beltrao P, Starita L et al (2013) Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods 10:676–682CrossRefPubMedGoogle Scholar
  59. 59.
    Iesmantavicius V, Weinert BT, Choudhary C (2014) Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells. Mol Cell Proteomics 13:1979–1992PubMedCentralCrossRefPubMedGoogle Scholar
  60. 60.
    Beltrao P, Bork P, Krogan NJ et al (2013) Evolution and functional cross-talk of protein post-translational modifications. Mol Syst Biol 9:714PubMedCentralCrossRefPubMedGoogle Scholar
  61. 61.
    Ficarro SB, McCleland ML, Stukenberg PT et al (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305CrossRefPubMedGoogle Scholar
  62. 62.
    Zubarev RA, Makarov A (2013) Orbitrap mass spectrometry. Anal Chem 85:5288–5296CrossRefPubMedGoogle Scholar
  63. 63.
    Hennrich ML, Groenewold V, Kops GJ, Heck AJ et al (2011) Improving depth in phosphoproteomics by using a strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach. Anal Chem 83:7137–7143CrossRefPubMedGoogle Scholar
  64. 64.
    Hennrich ML, van den Toorn HWP, Groenewold V et al (2012) Ultra acidic strong cation exchange enabling the efficient enrichment of basic phosphopeptides. Anal Chem 84:1804–1808CrossRefPubMedGoogle Scholar
  65. 65.
    Huttlin EL, Jedrychowski MP, Elias JE et al (2010) A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143:1174–1189PubMedCentralCrossRefPubMedGoogle Scholar
  66. 66.
    Lundby A, Secher A, Lage K et al (2012) Quantitative maps of protein phosphorlyation sites across 14 different rat organs and tissues. Nat Commun 3:876PubMedCentralCrossRefPubMedGoogle Scholar
  67. 67.
    Meijer LAT, Zhou H, Chan OYA et al (2013) Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway. Mol Bio Syst 9:732–749Google Scholar
  68. 68.
    Monetti M, Nagaraj N, Sharma K et al (2011) Large-scale phosphosite quantification in tissues by a spike-in SILAC method. Nat Methods 8:655–658CrossRefPubMedGoogle Scholar
  69. 69.
    Nagaraj N, D‘Souza RCJ, Cox J et al (2010) Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. J Proteome Res 9:6786–6794CrossRefPubMedGoogle Scholar
  70. 70.
    Olsen JV, Vermeulen M, Santamaria A et al (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3:ra3PubMedGoogle Scholar
  71. 71.
    Phanstiel DH, Brumbaugh J, Wenger CD et al (2011) Proteomic and phosphoproteomic comparison of human ES and iPS cells. Nat Methods 8:821–827PubMedCentralCrossRefPubMedGoogle Scholar
  72. 72.
    Rigbolt KTG, Prokhorova TA, Akimov V et al (2011) System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation. Sci Signal 4:rs3PubMedGoogle Scholar
  73. 73.
    Yi T, Zhai B, Yu Y et al (2014) Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells. Proc Natl Acad Sci U S A 111:E2182–E2190PubMedCentralCrossRefPubMedGoogle Scholar
  74. 74.
    Yue X-S, Hummon AB (2013) Combination of Multistep IMAC Enrichment with High-pH Reverse Phase Separation for In-Depth Phosphoproteomic Profiling. J Proteome Res 12:4176–4186CrossRefPubMedGoogle Scholar
  75. 75.
    Zarei M, Sprenger A, Gretzmeier C et al. (2012) Combinatorial Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) and Strong Cation Exchange (SCX) Chromatography for In-Depth Phosphoproteome Analysis. J Proteome Res 12:4269–4276CrossRefGoogle Scholar
  76. 76.
    Zhou H, Di Palma S, Preisinger C et al. (2013) Toward a Comprehensive Characterization of a Human Cancer Cell Phosphoproteome. J Proteome Res 13:260–271CrossRefGoogle Scholar
  77. 77.
    Huang J, Qin H, Dong J et al (2014) In Situ Sample Processing Approach (iSPA) for comprehensive quantitative phosphoproteome analysis. J Proteome Res 13:3896–3904CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of BiologyInstitute of Molecular Systems Biology, ETH ZurichZurichSwitzerland

Personalised recommendations