Advertisement

Analysis of Post-translational Modifications by LC-MS/MS

  • Hannah Johnson
  • Claire E. Eyers
Part of the Methods in Molecular Biology book series (MIMB, volume 658)

Abstract

Post-translational modifications are highly dynamic and known to regulate many cellular processes. Both the site and the stoichiometry of modification of a given protein sequence can have profound effects on the regulation of protein function. Thus, the identification of sites of post-translational modification is crucial for fully deciphering the biological roles of any given protein. The acute regulation and typically low stoichiometry of many post-translational modifications makes characterization of the sites of modification challenging. Thus, the development of analytical strategies to aid the selective enrichment and characterization of these species is paramount. Ongoing developments in mass spectrometry resulting in increased speed and sensitivity of analysis mean that mass spectrometry has become the ideal analytical tool for the qualitative and quantitative analysis of protein modifications. This chapter provides an overview of the most popular LC-MS/MS-based strategies for the enrichment of modified peptides/proteins and mass spectrometric workflows targeted toward the analysis of specific post-translationally modified analytes.

Key words

Post-translational modification mass spectrometry LC-MS/MS enrichment 

References

  1. 1.
    Walsh, C. (2005) Posttranslational modification of proteins: expanding nature’s inventory. B. Roberts, Colorado.Google Scholar
  2. 2.
    Hunter, T. (2000) Signaling―2000 and beyond. Cell 100, 113–127.PubMedCrossRefGoogle Scholar
  3. 3.
    Seet, B. T., Dikic, I., Zhou, M. M., and Pawson, T. (2006) Reading protein modifications with interaction domains. Nat. Rev. 7, 473–483.CrossRefGoogle Scholar
  4. 4.
    Eyers, C. E., and Gaskell, S. J. (2008) Mass spectrometry to identify post-translational modifications. Wiley Encyclopedia of Chemical Biology. doi:10.1002/9780470048672. wecb469.Google Scholar
  5. 5.
    Morelle, W., and Michalski, J. C. (2007) Analysis of protein glycosylation by mass spectrometry. Nat. Protoc 2, 1585–1602.PubMedCrossRefGoogle Scholar
  6. 6.
    Dwek, R. A. (1996) Glycobiology: toward understanding the function of sugars. Chem. Rev. 96, 683–720.PubMedCrossRefGoogle Scholar
  7. 7.
    Helenius, A., and Aebi, M. (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019–1049.PubMedCrossRefGoogle Scholar
  8. 8.
    Weissman, A. M. (2001) Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell. Biol. 2, 169–178.PubMedCrossRefGoogle Scholar
  9. 9.
    Drews, O., Zong, C., and Ping, P. (2007) Exploring proteasome complexes by proteomic approaches. Proteomics 7, 1047–1058.PubMedCrossRefGoogle Scholar
  10. 10.
    Washburn, M. P., Wolters, D., and Yates, J. R., 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  11. 11.
    Haydon, C. E., Eyers, P. A., Aveline-Wolf, L. D., Resing, K. A., Maller, J. L., and Ahn, N. G. (2003) Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography. Mol. Cell. Proteomics 2, 1055–1067.PubMedCrossRefGoogle Scholar
  12. 12.
    Nuhse, T. S., Stensballe, A., Jensen, O. N., and Peck, S. C. (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 2, 1234–1243.PubMedCrossRefGoogle Scholar
  13. 13.
    Reinders, J., and Sickmann, A. (2005) State-of-the-art in phosphoproteomics. Proteomics 5, 4052–4061.PubMedCrossRefGoogle Scholar
  14. 14.
    Schweppe, R. E., Haydon, C. E., Lewis, T. S., Resing, K. A., and Ahn, N. G. (2003) The characterization of protein post-translational modifications by mass spectrometry. Acc. Chem, Res. 36, 453–461.CrossRefGoogle Scholar
  15. 15.
    Fraga, M. F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G., Bonaldi, T., Haydon, C., Ropero, S., Petrie, K., Iyer, N. G., Perez-Rosado, A., Calvo, E., Lopez, J. A., Cano, A., Calasanz, M. J., Colomer, D., Piris, M. A., Ahn, N., Imhof, A., Caldas, C., Jenuwein, T., and Esteller, M. (2005) Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37, 391–400.PubMedCrossRefGoogle Scholar
  16. 16.
    Tyler, R. K., Chu, M. L., Johnson, H., McKenzie, E. A., Gaskell, S. J., and Eyers, P. A. (2009) Phosphoregulation of human Mps1 kinase. Biochem. J. 417, 173–181.PubMedCrossRefGoogle Scholar
  17. 17.
    Mirgorodskaya, E., Roepstorff, P., and Zubarev, R. A. (1999) Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer. Anal. Chem. 71, 4431–4436.PubMedCrossRefGoogle Scholar
  18. 18.
    Zubarev, R. A. (2004) Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 15, 12–16.PubMedCrossRefGoogle Scholar
  19. 19.
    Tsybin, Y. O., Ramstrom, M., Witt, M., Baykut, G., and Hakansson, P. (2004) Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry. J. Mass Spectrom. 39, 719–729.PubMedCrossRefGoogle Scholar
  20. 20.
    Swaney, D. L., McAlister, G. C., Wirtala, M., Schwartz, J. C., Syka, J. E., and Coon, J. J. (2007) Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485.PubMedCrossRefGoogle Scholar
  21. 21.
    Swaney, D. L., McAlister, G. C., Wirtala, M., Schwartz, J. C., Syka, J. E. P., and Coon, J. J. (2007) Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485.PubMedCrossRefGoogle Scholar
  22. 22.
    Chi, A., Huttenhower, C., Geer, L. Y., Coon, J. J., Syka, J. E. P., Bai, D. L., Shabanowitz, J., Burke, D. J., Troyanskaya, O. G., and Hunt, D. F. (2007) Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. 10.1073/pnas.0607084104. Proc. Natl. Acad. Sci. USA 104, 2193–2198.PubMedCrossRefGoogle Scholar
  23. 23.
    Zhang, Q., Schepmoes, A. A., Brock, J. W. C., Wu, S., Moore, R. J., Purvine, S. O., Baynes, J. W., Smith, R. D., and Metz, T. O. (2008) Improved methods for the enrichment and analysis of glycated peptides. doi:10.1021/ac801704j. Anal. Chem. 80, 9822–9829.PubMedCrossRefGoogle Scholar
  24. 24.
    Medzihradszky, K. F., Guan, S., Maltby, D. A., and Burlingame, A. L. (2007) Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation. J. Am. Soc. Mass Spectrom. 18, 1617–1624.PubMedCrossRefGoogle Scholar
  25. 25.
    Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., and Kirschner, M. W. (2006) Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 5, 172–181.PubMedGoogle Scholar
  26. 26.
    Peng, J., Schwartz, D., Elias, J. E., Thoreen, C. C., Cheng, D., Marsischky, G., Roelofs, J., Finley, D., and Gygi, S. P. (2003) A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926.PubMedCrossRefGoogle Scholar
  27. 27.
    Blagoev, B., Ong, S.-E., Kratchmarova, I., and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139–1145.PubMedCrossRefGoogle Scholar
  28. 28.
    Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., Zhang, H., Zha, X. M., Polakiewicz, R. D., and Comb, M. J. (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101.PubMedCrossRefGoogle Scholar
  29. 29.
    Cortez, D., Glick, G., and Elledge, S. J. (2004) Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc. Natl. Acad. Sci. USA 101, 10078–10083.PubMedCrossRefGoogle Scholar
  30. 30.
    Gronborg, M., Kristiansen, T. Z., Stensballe, A., Andersen, J. S., Ohara, O., Mann, M., Jensen, O. N., and Pandey, A. (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol. Cell. Proteomics 1, 517–527.PubMedCrossRefGoogle Scholar
  31. 31.
    Kane, S., Sano, H., Liu, S. C., Asara, J. M., Lane, W. S., Garner, C. C., and Lienhard, G. E. (2002) A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J. Biol. Chem. 277, 22115–22118.PubMedCrossRefGoogle Scholar
  32. 32.
    Vasilescu, J., Smith, J. C., Ethier, M., and Figeys, D. (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. doi:10.1021/pr050265i. J. Proteome Res. 4, 2192–2200.PubMedCrossRefGoogle Scholar
  33. 33.
    Nawarak, J., Phutrakul, S., and Chen, S.-T. (2004) Analysis of lectin-bound glycoproteins in snake venom from the Elapidae and Viperidae families. J. Proteome Res. 3, 383–392.PubMedCrossRefGoogle Scholar
  34. 34.
    Qiu, R., and Regnier, F. E. (2005) Use of Multidimensional lectin affinity chromatography in differential glycoproteomics. doi:10.1021/ac048751x. Anal. Chem. 77, 2802–2809.PubMedCrossRefGoogle Scholar
  35. 35.
    Drake, R. R., Schwegler, E. E., Malik, G., Diaz, J., Block, T., Mehta, A., and Semmes, O. J. (2006) Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers. 10.1074/mcp.M600176-MCP200. Mol. Cell. Proteomics 5, 1957–1967.PubMedCrossRefGoogle Scholar
  36. 36.
    Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 154, 250–254.PubMedCrossRefGoogle Scholar
  37. 37.
    Posewitz, M. C., and Tempst, P. (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides. doi:10.1021/ac981409y. Anal. Chem. 71, 2883–2892.PubMedCrossRefGoogle Scholar
  38. 38.
    Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. D. (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. 10.1074/mcp.T500007-MCP200. Mol. Cell. Proteomics 4, 873–886.PubMedCrossRefGoogle Scholar
  39. 39.
    Kweon, H. K., and Hakansson, K. (2006) Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. doi:10.1021/ac0522355. Anal. Chem. 78, 1743–1749.PubMedCrossRefGoogle Scholar
  40. 40.
    Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305.PubMedCrossRefGoogle Scholar
  41. 41.
    Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886.PubMedCrossRefGoogle Scholar
  42. 42.
    Wu, J., Shakey, Q., Liu, W., Schuller, A., and Follettie, M. T. (2007) Global profiling of phosphopeptides by titania affinity enrichment. J. Proteome Res. 6, 4684–4689.PubMedCrossRefGoogle Scholar
  43. 43.
    Thingholm, T. E., Jensen, O. N., Robinson, P. J., and Larsen, M. R. (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol. Cell. Proteomics 7, 661–671.PubMedGoogle Scholar
  44. 44.
    Jensen, S. S., and Larsen, M. R. (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun. Mass Spectrom. 21, 3635–3645.PubMedCrossRefGoogle Scholar
  45. 45.
    Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135.PubMedCrossRefGoogle Scholar
  46. 46.
    Han, G., Ye, M., Zhou, H., Jiang, X., Feng, S., Tian, R., Wan, D., Zou, H., and Gu, J. (2008) Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography. Proteomics 8, 1346–1361.PubMedCrossRefGoogle Scholar
  47. 47.
    Zhang, X., Ye, J., Jensen, O. N., and Roepstorff, P. (2007) Highly efficient phosphopeptide enrichment by calcium phosphate precipitation combined with subsequent IMAC enrichment. Mol. Cell. Proteomics 6, 2032–2042.PubMedCrossRefGoogle Scholar
  48. 48.
    Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N., and Roepstorff, P. (2004) A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J. Proteome Res. 3, 556–566.PubMedCrossRefGoogle Scholar
  49. 49.
    Calvano, C. D., Zambonin, C. G., and Jensen, O. N. (2008) Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry. J. Proteomics 71, 304–317.PubMedCrossRefGoogle Scholar
  50. 50.
    Picariello, G., Ferranti, P., Mamone, G., Roepstorff, P., and Addeo, F. (2008) Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry. Proteomics 8, 3833–3847.PubMedCrossRefGoogle Scholar
  51. 51.
    Wuhrer, M., de Boer, A. R., and Deelder, A. M. (2009) Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry. Mass Spectrom. Rev. 28, 192–206.Google Scholar
  52. 52.
    IUPAC (1997) Compendium of chemical terminology, 2nd (The “Gold Book”) ed, Blackwell Scientific Publications, Oxford.Google Scholar
  53. 53.
    Carr, S. A., Huddleston, M. J., and Annan, R. S. (1996) Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal. Biochem. 239, 180–192.PubMedCrossRefGoogle Scholar
  54. 54.
    Steen, H., Pandey, A., Andersen, J. S., and Mann, M. (2002) Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method. 10.1126/stke.2002.154.pl16. Sci. STKE 2002, pl16.CrossRefGoogle Scholar
  55. 55.
    Huddleston, M. J., Bean, M. F., and Carr, S. A. (1993) Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal. Chem. 65, 877–884.PubMedCrossRefGoogle Scholar
  56. 56.
    Bean, M. F., Annan, R. S., Hemling, M. E., Mentzer, M., Huddleston, M. J., and Carr, S. A. (1995) LC-MS methods for selective detection of posttranslational modifications in proteins: glycosylation, phosphorylation, sulfation, and acylation techniques in protein chemistry. In Crabb, J. W. (Ed.), Vol. 6, pp. 107–116, Academic Press.Google Scholar
  57. 57.
    Kim, J. Y., Kim, K. W., Kwon, H. J., Lee, D. W., and Yoo, J. S. (2002) Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. doi:10.1021/ac0256080. Anal. Chem. 74, 5443–5449.PubMedCrossRefGoogle Scholar
  58. 58.
    Sweet, S. M., Mardakheh, F. K., Ryan, K. J., Langton, A. J., Heath, J. K., and Cooper, H. J. (2008) Targeted online liquid chromatography electron capture dissociation mass spectrometry for the localization of sites of in vivo phosphorylation in human Sprouty2. Anal. Chem. 80, 6650–6657.PubMedCrossRefGoogle Scholar
  59. 59.
    Zubarev, R. A., Zubarev, A. R., and Savitski, M. M. (2008) Electron capture/transfer versus collisionally activated/induced dissociations: solo or duet? J. Am. Soc. Mass Spectrom. 19, 753–761.PubMedCrossRefGoogle Scholar
  60. 60.
    Unwin, R. D., Griffiths, J. R., Leverentz, M. K., Grallert, A., Hagan, I. M., and Whetton, A. D. (2005) Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. 10.1074/mcp.M500113-MCP200. Mol. Cell. Proteomics 4, 1134–1144.PubMedCrossRefGoogle Scholar
  61. 61.
    Sahana Mollah, I. E. W., Phung, Q., Arnott, D., Dixit, V. M., Lill, J. R. (2007) Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. Rapid Commun. Mass Spectrom. 21, 3357–3364.PubMedCrossRefGoogle Scholar
  62. 62.
    Hegeman, A. D., Harms, A. C., Sussman, M. R., Bunner, A. E., and Harper, J. F. (2004) An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins. J. Am. Soc. Mass Spectrom. 15, 647–653.PubMedCrossRefGoogle Scholar
  63. 63.
    Zhang, X., Jin, Q. K., Carr, S. A., and Annan, R. S. (2002) N-terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry. Rapid Commun. Mass Spectrom. 16, 2325–2332.PubMedCrossRefGoogle Scholar
  64. 64.
    Mayor, T., Graumann, J., Bryan, J., MacCoss, M. J., and Deshaies, R. J. (2007) Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol. Cell. Proteomics 6, 1885–1895.PubMedCrossRefGoogle Scholar
  65. 65.
    Mayya, V., Rezual, K., Wu, L., Fong, M. B., and Han, D. K. (2006) Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases. Mol. Cell. Proteomics 5, 1146–1157.PubMedCrossRefGoogle Scholar
  66. 66.
    Bonenfant, D., Towbin, H., Coulot, M., Schindler, P., Mueller, D. R., and van Oostrum, J. (2007) Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry. Mol. Cell. Proteomics 6, 1917–1932.PubMedCrossRefGoogle Scholar
  67. 67.
    Pan, C., Gnad, F., Olsen, J. V., and Mann, M. (2008) Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors. Proteomics 8, 4534–4546.PubMedCrossRefGoogle Scholar
  68. 68.
    Amanchy, R., Kalume, D. E., and Pandey, A. (2005) Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications. Sci STKE 2005, pl2.CrossRefGoogle Scholar
  69. 69.
    Steen, H., Jebanathirajah, J. A., Springer, M., and Kirschner, M. W. (2005) Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc. Natl. Acad. Sci. USA 102, 3948–3953.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hannah Johnson
    • 1
  • Claire E. Eyers
    • 1
  1. 1.Michael Barber Centre for Mass Spectrometry, School of ChemistryThe University of ManchesterManchesterUK

Personalised recommendations