Interpreting the protein language using proteomics

Abstract

Post-translational modifications define the functional and structural plasticity of proteins in archaea, prokaryotes and eukaryotes. Multi-site protein modification modulates protein activity and macromolecular interactions and is involved in a range of fundamental molecular processes. Combining state-of-the-art technologies in molecular cell biology, protein mass spectrometry and bioinformatics, it is now feasible to discover and study the structural and functional roles of distinct protein post-translational modifications.

Key Points

  • Post-translational modifications (PTMs) affect protein conformation and interactions and can mediate the sequestration of proteins to cellular compartments and organelles.

  • The integration of molecular-cell-biology, protein-mass-spectrometry and bioinformatics technologies facilitates the study of protein PTMs.

  • Mass spectrometry identifies PTMs by looking for distinct mass increments or diagnostic PTM-specific signals that are generated by the tandem-mass-spectrometry analysis of peptides and proteins.

  • Modification-specific proteomics takes advantage of organelle purification and PTM-specific affinity-enrichment methods, multidimensional peptide-separation methods and tandem mass spectrometry to achieve high sensitivity and selectivity for the determination of PTMs.

  • The quantitative analysis of PTMs is possible. This can be achieved by using two-dimensional gel-based methods and imaging or by using liquid-chromatography–mass-spectrometry together with peptide-intensity-profiling or stable-isotope-labelling approaches.

  • Functional phosphoproteomics studies are delineating cellular signal-transduction cascades and revealing new regulatory mechanisms in microbes, plants and mammals.

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Figure 1: Cellular post-translational modifications.
Figure 2: Mechanism of action of post-translational modifications.

References

  1. 1

    Krishna, R. G. & Wold, F. Post-translational modification of proteins. Adv. Enzymol. Relat. Areas Mol. Biol. 67, 265–298 (1993).

  2. 2

    Jensen, O. N. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Curr. Opin. Chem. Biol. 8, 33–41 (2004).

  3. 3

    Mann, M. & Jensen, O. N. Proteomic analysis of post-translational modifications. Nature Biotechnol. 21, 255–261 (2003).

  4. 4

    Eichler, J. & Adams, M. W. Posttranslational protein modification in Archaea. Microbiol. Mol. Biol. Rev. 69, 393–425 (2005).

  5. 5

    Yang, X. J. Multisite protein modification and intramolecular signaling. Oncogene 24, 1653–1662 (2005). An excellent review of multi-site PTMs and their biological roles.

  6. 6

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

  7. 7

    Gunawardena, J. Multisite protein phosphorylation makes a good threshold but can be a poor switch. Proc. Natl Acad. Sci. USA 102, 14617–14622 (2005).

  8. 8

    Cosgrove, M. S., Boeke, J. D. & Wolberger, C. Regulated nucleosome mobility and the histone code. Nature Struct. Mol. Biol. 11, 1037–1043 (2004).

  9. 9

    Fischle, W., Wang, Y. & Allis, C. D. Binary switches and modification cassettes in histone biology and beyond. Nature 425, 475–479 (2003).

  10. 10

    Pokholok, D. K. et al. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517–527 (2005).

  11. 11

    Fischle, W. et al. Regulation of HP1–chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122 (2005).

  12. 12

    Freitas, M. A., Sklenar, A. R. & Parthun, M. R. Application of mass spectrometry to the identification and quantification of histone post-translational modifications. J. Cell. Biochem. 92, 691–700 (2004).

  13. 13

    Insinga, A., Minucci, S. & Pelicci, P. G. Mechanisms of selective anticancer action of histone deacetylase inhibitors. Cell Cycle 4, 741–743 (2005).

  14. 14

    Steen, H. & Mann, M. The abc's (and xyz's) of peptide sequencing. Nature Rev. Mol. Cell Biol. 5, 699–711 (2004). A good introduction to the concepts and methods that are involved in peptide sequencing by MS/MS.

  15. 15

    Yates, J. R. 3rd, Gilchrist, A., Howell, K. E. & Bergeron, J. J. Proteomics of organelles and large cellular structures. Nature Rev. Mol. Cell Biol. 6, 702–714 (2005).

  16. 16

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

  17. 17

    Loughrey Chen, S. et al. Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast. Mol. Cell. Proteomics 1, 186–196 (2002).

  18. 18

    Neubauer, G. & Mann, M. Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal. Chem. 71, 235–242 (1999).

  19. 19

    Unwin, R. D. et al. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol. Cell. Proteomics 4, 1134–1144 (2005).

  20. 20

    Beausoleil, S. A. et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl Acad. Sci. USA 101, 12130–12135 (2004).

  21. 21

    Gruhler, A. et al. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol. Cell. Proteomics 4, 310–327 (2005). A large-scale quantitative phosphoproteomics study using affinity enrichment and multi-stage MS reveals some of the key signalling modules that are involved in the yeast pheromone response.

  22. 22

    Coon, J. J. et al. Protein identification using sequential ion–ion reactions and tandem mass spectrometry. Proc. Natl Acad. Sci. USA 102, 9463–9468 (2005).

  23. 23

    Sze, S. K., Ge, Y., Oh, H. & McLafferty, F. W. Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue. Proc. Natl Acad. Sci. USA 99, 1774–1779 (2002).

  24. 24

    Wu, S. L., Kim, J., Hancock, W. S. & Karger, B. Extended range proteomic analysis (ERPA): a new and sensitive LC–MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of β-casein and epidermal growth factor receptor (EGFR). J. Proteome Res. 4, 1155–1170 (2005).

  25. 25

    Creasy, D. M. & Cottrell, J. S. Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2, 1426–1434 (2002).

  26. 26

    Matthiesen, R., Trelle, M. B., Højrup, P., Bunkenborg, J. & Jensen, O. N. VEMS 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins. J. Proteome Res. 4, 2327–2337 (2005).

  27. 27

    Sadygov, R. G., Cociorva, D. & Yates, J. R. 3rd. Large-scale database searching using tandem mass spectra: looking up the answer in the back of the book. Nature Methods 1, 195–202 (2004).

  28. 28

    Cantin, G. T. & Yates, J. R. 3rd. Strategies for shotgun identification of post-translational modifications by mass spectrometry. J. Chromatogr. A 1053, 7–14 (2004).

  29. 29

    Kirkpatrick, D. S., Denison, C. & Gygi, S. P. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nature Cell Biol. 7, 750–757 (2005). Excellent review on proteomics approaches for the study of ubiquitylation and protein modification by ubiquitin-like modifiers.

  30. 30

    Boeri Erba, E. et al. Systematic analysis of the epidermal growth factor receptor by mass spectrometry reveals stimulation-dependent multisite phosphorylation. Mol. Cell. Proteomics 4, 1107–1121 (2005).

  31. 31

    Pandey, A. et al. Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl Acad. Sci. USA 97, 179–184 (2000).

  32. 32

    Salomon, A. R. et al. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl Acad. Sci. USA 100, 443–448 (2003).

  33. 33

    Blagoev, B., Ong, S. E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnol. 22, 1139–1145 (2004). A temporal analysis that used triplex stable-isotope labelling and MS to study EGF-induced phosphotyrosine signalling events.

  34. 34

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

  35. 35

    Gronborg, M. et al. 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 (2002).

  36. 36

    Kane, S. et al. 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 (2002).

  37. 37

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

  38. 38

    Stensballe, A., Andersen, S. & Jensen, O. N. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 1, 207–222 (2001).

  39. 39

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

  40. 40

    Nuhse, T. S., Stensballe, A., Jensen, O. N. & Peck, S. C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 2, 1234–1243 (2003).

  41. 41

    Zhang, Y. et al. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol. Cell. Proteomics 4, 1240–1250 (2005).

  42. 42

    Collins, M. O. et al. Proteomic analysis of in vivo phosphorylated synaptic proteins. J. Biol. Chem. 280, 5972–5982 (2005).

  43. 43

    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).

  44. 44

    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).

  45. 45

    Dube, D. H. & Bertozzi, C. R. Glycans in cancer and inflammation — potential for therapeutics and diagnostics. Nature Rev. Drug Discov. 4, 477–488 (2005).

  46. 46

    Wilson, N. L., Schulz, B. L., Karlsson, N. G. & Packer, N. H. Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins. J. Proteome Res. 1, 521–529 (2002).

  47. 47

    Harvey, D. J. Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry. Expert Rev. Proteomics 2, 87–101 (2005). A comprehensive overview of MS-based methods for glycoprotein and glycan analysis using proteomics approaches.

  48. 48

    Gabius, H. J., Andre, S., Kaltner, H. & Siebert, H. C. The sugar code: functional lectinomics. Biochim. Biophys. Acta 1572, 165–177 (2002).

  49. 49

    Yang, Z. & Hancock, W. S. Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column. J. Chromatogr. A 1053, 79–88 (2004).

  50. 50

    Kaji, H. et al. Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nature Biotechnol. 21, 667–672 (2003).

  51. 51

    Küster, B. & Mann, M. 18O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching. Anal. Chem. 71, 1431–1440 (1999).

  52. 52

    Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N. & Roepstorff, P. 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 (2004).

  53. 53

    Küster, B., Krogh, T. N., Mortz, E. & Harvey, D. J. Glycosylation analysis of gel-separated proteins. Proteomics 1, 350–361 (2001).

  54. 54

    Larsen, M. R., Hojrup, P. & Roepstorff, P. Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry. Mol. Cell. Proteomics 4, 107–119 (2005).

  55. 55

    McLachlin, D. T. & Chait, B. T. Improved β-elimination-based affinity purification strategy for enrichment of phosphopeptides. Anal. Chem. 75, 6826–6836 (2003).

  56. 56

    Zhou, H., Watts, J. D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nature Biotechnol. 19, 375–378 (2001).

  57. 57

    Tao, W. A. et al. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nature Methods 2, 591–598 (2005).

  58. 58

    Brittain, S. M., Ficarro, S. B., Brock, A. & Peters, E. C. Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry. Nature Biotechnol. 23, 463–468 (2005).

  59. 59

    Kho, Y. et al. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl Acad. Sci. USA 101, 12479–12484 (2004).

  60. 60

    Sprung, R. et al. Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J. Proteome Res. 4, 950–957 (2005).

  61. 61

    Gevaert, K. et al. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nature Biotechnol. 21, 566–569 (2003).

  62. 62

    Van Damme, P. et al. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nature Methods 2, 771–777 (2005).

  63. 63

    Welchman, R. L., Gordon, C. & Mayer, R. J. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nature Rev. Mol. Cell Biol. 6, 599–609 (2005).

  64. 64

    Giannakopoulos, N. V. et al. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. Commun. 336, 496–506 (2005).

  65. 65

    Ghezzi, P. & Bonetto, V. Redox proteomics: identification of oxidatively modified proteins. Proteomics 3, 1145–1153 (2003).

  66. 66

    Aulak, K. S., Koeck, T., Crabb, J. W. & Stuehr, D. J. Proteomic method for identification of tyrosine-nitrated proteins. Methods Mol. Biol. 279, 151–165 (2004).

  67. 67

    Kanski, J. & Schoneich, C. Protein nitration in biological aging: proteomic and tandem mass spectrometric characterization of nitrated sites. Methods Enzymol. 396, 160–171 (2005).

  68. 68

    Miyagi, M. et al. Evidence that light modulates protein nitration in rat retina. Mol. Cell. Proteomics 1, 293–303 (2002).

  69. 69

    Kanski, J., Hong, S. J. & Schoneich, C. Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry. J. Biol. Chem. 280, 24261–24266 (2005).

  70. 70

    MacCoss, M. J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl Acad. Sci. USA 99, 7900–7905 (2002).

  71. 71

    Wu, C. C., MacCoss, M. J., Howell, K. E. & Yates, J. R. 3rd. A method for the comprehensive proteomic analysis of membrane proteins. Nature Biotechnol. 21, 532–538 (2003).

  72. 72

    Elortza, F. et al. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins. Mol. Cell. Proteomics 2, 1261–1270 (2003).

  73. 73

    Steen, H., Jebanathirajah, J. A., Springer, M. & Kirschner, M. W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc. Natl Acad. Sci. USA 102, 3948–3953 (2005).

  74. 74

    Oda, Y., Huang, K., Cross, F. R., Cowburn, D. & Chait, B. T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl Acad. Sci. USA 96, 6591–6596 (1999).

  75. 75

    Heck, A. J. & Krijgsveld, J. Mass spectrometry-based quantitative proteomics. Expert Rev. Proteomics 1, 317–326 (2004).

  76. 76

    Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W. & Gygi, S. P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl Acad. Sci. USA 100, 6940–6945 (2003).

  77. 77

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

  78. 78

    Krijgsveld, J. et al. Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nature Biotechnol. 21, 927–931 (2003).

  79. 79

    Gruhler, A., Schulze, W. X., Matthiesen, R., Mann, M. & Jensen, O. N. Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry. Mol. Cell. Proteomics 4, 1697–1709 (2005).

  80. 80

    Ibarrola, N., Molina, H., Iwahori, A. & Pandey, A. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C] tyrosine. J. Biol. Chem. 279, 15805–15813 (2004).

  81. 81

    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).

  82. 82

    Zhu, H., Pan, S., Gu, S., Bradbury, E. M. & Chen, X. Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Commun. Mass Spectrom. 16, 2115–2123 (2002).

  83. 83

    Kratchmarova, I., Blagoev, B., Haack-Sorensen, M., Kassem, M. & Mann, M. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science 308, 1472–1477 (2005).

  84. 84

    Ballif, B. A. et al. Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proc. Natl Acad. Sci. USA 102, 667–672 (2005).

  85. 85

    Guo, D. et al. A tethered catalysis, two-hybrid system to identify protein–protein interactions requiring post-translational modifications. Nature Biotechnol. 22, 888–892 (2004).

  86. 86

    Ptacek, J. et al. Global analysis of protein phosphorylation in yeast. Nature 438, 679–684 (2005).

  87. 87

    Gorg, A., Weiss, W. & Dunn, M. J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685 (2004). An overview of 2D-gel electrophoresis, which includes its application to the study of protein diversity and PTMs.

  88. 88

    Ge, Y. et al. Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness. Proteomics 4, 3464–3467 (2004).

  89. 89

    Meng, F., Forbes, A. J., Miller, L. M. & Kelleher, N. L. Detection and localization of protein modifications by high resolution tandem mass spectrometry. Mass Spectrom. Rev. 24, 126–134 (2005).

  90. 90

    Hu, Q. et al. The Orbitrap: a new mass spectrometer. J. Mass Spectrom. 40, 430–443 (2005).

  91. 91

    Uhlen, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell. Proteomics 4, 1920–1932 (2005).

  92. 92

    Kelleher, N. L. et al. Localization of labile posttranslational modifications by electron capture dissociation: the case of γ-carboxyglutamic acid. Anal. Chem. 71, 4250–4253 (1999).

  93. 93

    Zubarev, R. A. Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 15, 12–16 (2004).

  94. 94

    Syka, J. E., Coon, J. J., Schroeder, M. J., Shabanowitz, J. & Hunt, D. F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl Acad. Sci. USA 101, 9528–9533 (2004).

  95. 95

    Forbes, A. J. et al. Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS. Proc. Natl Acad. Sci. USA 101, 2678–2683 (2004).

  96. 96

    Zabrouskov, V., Giacomelli, L., van Wijk, K. J. & McLafferty, F. W. A new approach for plant proteomics: characterization of chloroplast proteins of Arabidopsis thaliana by top-down mass spectrometry. Mol. Cell. Proteomics 2, 1253–1260 (2003).

  97. 97

    Jones, P. et al. PRIDE: a public repository of protein and peptide identifications for the proteomics community. Nucleic Acids Res. 34, D659–D663 (2006).

  98. 98

    Orchard, S., Hermjakob, H., Taylor, C., Aebersold, R. & Apweiler, R. Human proteome organisation proteomics standards initiative pre-congress initiative. Proteomics 5, 4651–4652 (2005).

  99. 99

    Peri, S. et al. Development of human protein reference database as an initial platform for approaching systems biology in humans. Genome Res. 13, 2363–2371 (2003).

  100. 100

    Blom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S. & Brunak, S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649 (2004).

  101. 101

    Chang, E. J., Archambault, V., McLachlin, D. T., Krutchinsky, A. N. & Chait, B. T. Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry. Anal. Chem. 76, 4472–4483 (2004).

  102. 102

    Hjerrild, M. et al. Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry. J. Proteome Res. 3, 426–433 (2004).

  103. 103

    Nuhse, T. S., Stensballe, A., Jensen, O. N. & Peck, S. C. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16, 2394–2405 (2004).

  104. 104

    Schwartz, D. & Gygi, S. P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nature Biotechnol. 23, 1391–1398 (2005).

  105. 105

    de Lichtenberg, U., Jensen, L. J., Brunak, S. & Bork, P. Dynamic complex formation during the yeast cell cycle. Science 307, 724–727 (2005). Shows the power of an integrative computational analysis of gene-expression data and protein–protein interaction data for the characterization of dynamic, functional protein modules.

  106. 106

    Rual, J. F. et al. Towards a proteome-scale map of the human protein–protein interaction network. Nature 437, 1173–1178 (2005).

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Acknowledgements

I thank my colleagues in the Protein Research Group, as well as numerous colleagues in the world of proteomics, for many interesting and stimulating scientific discussions. I am grateful to M. B. Trelle for his assistance in preparing the figures. This manuscript is dedicated to the memory of my late father, Alex B. Jensen (1940–2005).

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FURTHER INFORMATION

Center for Biological Sequence Analysis Prediction Servers (Technical University of Denmark)

Human Protein Reference Database (HPRD)

HUPO Proteomics Standards Initiative

PhosphoELM (a database of S/T/Y phosphorylation sites)

Protein Research Group, University of Southern Denmark

The Association of Biomolecular Resource Facilities

UNIMOD (protein modifications for mass spectrometry)

Glossary

ChIP-on-chip

A chromatin immunoprecipitation (ChIP) experiment that uses protein–DNA crosslinking, specific antibodies and cDNA microarrays ('chips') for large-scale studies of DNA-binding proteins, including the analysis of post-translational modifications.

Microfluidics

Nanolitre-flow chromatographic systems for protein and peptide separations prior to mass-spectrometry analysis.

Electrospray ionization

A 'soft' ionization method for mass spectrometry that generates gas-phase ions from peptide and protein solutions through the vapourization of liquid in an electric field.

Matrix-assisted laser desorption/ionization

A 'soft' ionization technique for mass spectrometry that produces gas-phase ions through the pulsed, ultraviolet laser irradiation of crystalline deposits of peptides and proteins.

TiO2 columns

Titanium dioxide interacts with phosphate groups by a chelation mechanism. These columns are therefore useful for the selective and specific enrichment of phosphopeptides.

β-elimination reaction

The alkaline-induced elimination of phosphoric acid from phosphoserine and phosphothreonine residues or of O-linked sugars from O-glycosylated residues in peptides.

Michael addition reaction

The addition of a functional chemical moiety to a residue that was post-translationally modified but that has undergone a β-elimination reaction. Affinity tags or mass tags can be added to allow the specific recovery or detection of post-translationally modified peptides.

Phosphoramidate chemistry

A method for the selective capture of converted post-translationally modified peptides, with the aim of identifying the modified protein and the site of the modification.

Chromatographic stationary phases

The column resins that are used for the chromatographic separation of molecules — for example, a reversed-phase resin or a strong-cation-exchange resin.

Cell-signalling cassettes

Multiprotein modules that are involved in signal-transduction processes and that rapidly propagate stimuli from cell-surface receptors to the cytosol or the nucleus.

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