Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Activity-Based Protein Profiling

Applications to Biomarker Discovery, In Vivo Imaging and Drug Discovery

Abstract

The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them.

The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states. In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity.

Chemical probes can be ‘tuned’ to react with defined enzymatic targets through the use of chemically reactive warhead groups, fused to selective binding elements that control their overall specificity. As a result, ABPs function as highly specific, mechanism-based reagents that provide a direct readout of enzymatic activity within complex proteomes. Modification of protein targets by an ABP facilitates their purification and isolation, thereby eliminating many of the confounding issues of dynamic range in protein abundance. In this review, we outline recent advances in the field of chemical proteomics. Specifically, we highlight how this technology can be applied to advance the fields of biomarker discovery, in vivo imaging, and small molecule screening and drug target discovery.

This is a preview of subscription content, log in to check access.

Fig. 1
Table I
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Venter JC, Adams MD, Myers EW, et al. The sequence of the human genome. Science 2001 Feb 16; 291: 1304–51

  2. 2.

    Corthals GL, Wasinger VC, Hochstrasser DF, et al. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000 Apr; 21: 1104–15

  3. 3.

    Patton WF. A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 2000 Apr; 21: 1123–44

  4. 4.

    Santoni V, Molloy M, Rabilloud T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000 Apr; 21: 1054–70

  5. 5.

    Gygi SP, Corthals GL, Zhang Y, et al. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 2000 Aug 15; 97: 9390–5

  6. 6.

    Gygi SP, Rist B, Gerber SA, et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999 Oct; 17: 994–9

  7. 7.

    Zhou H, Ranish JA, Watts JD, et al. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 2002 May; 20: 512–5

  8. 8.

    Goshe MB, Smith RD. Stable isotope-coded proteomic mass spectrometry. Curr Opin Biotechnol 2003 Feb; 14: 101–9

  9. 9.

    Petricoin EF, Liotta LA. SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer. Curr Opin Biotechnol 2004 Feb; 15: 24–30

  10. 10.

    Jeffery DA, Bogyo M. Chemical proteomics and its application to drug discovery. Curr Opin Biotechnol 2003 Feb; 14: 87–95

  11. 11.

    Speers AE, Cravatt BF. Chemical strategies for activity-based proteomics. Chembiochem 2004 Jan 3; 5: 41–7

  12. 12.

    Adam GC, Cravatt BF, Sorensen EJ. Profiling the specific reactivity of the proteome with non-directed activity-based probes. Chem Biol 2001 Jan; 8: 81–95

  13. 13.

    Adam GC, Sorensen EJ, Cravatt BF. Chemical strategies for functional proteomics. Mol Cell Proteomics 2002 Oct; 1: 781–90

  14. 14.

    Greenbaum D, Medzihradszky KF, Burlingame A, et al. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol 2000 Aug; 7: 569–81

  15. 15.

    Greenbaum D, Baruch A, Hayrapetian L, et al. Chemical approaches for functionally probing the proteome. Mol Cell Proteomics 2002 Jan; 1: 60–8

  16. 16.

    Mason RW, Bartholomew LT, Hardwick BS. The use of benzyloxy-carbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues. Biochem J 1989 Nov 1; 263: 945–9

  17. 17.

    Shi GP, Munger JS, Meara JP, et al. Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease. J Biol Chem 1992; 267(11): 7258–62

  18. 18.

    Bogyo M, Verhelst S, Bellingard-Dubouchaud V, et al. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem Biol 2000 Jan; 7: 27–38

  19. 19.

    Schaschke N, Assfalg-Machleidt I, Lassleben T, et al. Epoxysuccinyl peptide-derived affinity labels for cathepsin B. FEBS Lett 2000; 482(1–2): 91–6

  20. 20.

    Borodovsky A, Kessler BM, Casagrande R, et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 2001; 20(18): 5187–96

  21. 21.

    Mikolajczyk J, Boatright KM, Stennicke HR, et al. Sequential autolytic processing activates the zymogen of Arg-gingipain. J Biol Chem 2003; 278(12): 10458–64

  22. 22.

    Liu Y, Patricelli MP, Cravatt BF. Activity-based protein profiling: the serine hydrolases. Proc Natl Acad Sci U S A 1999 Dec 21; 96: 14694–9

  23. 23.

    Patricelli MP, Giang DK, Stamp LM, et al. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 2001 Sep; 1: 1067–71

  24. 24.

    Kidd D, Liu Y, Cravatt BF. Profiling serine hydrolase activities in complex proteomes. Biochemistry 2001; 40(13): 4005–15

  25. 25.

    Meng L, Kwok BH, Sin N, et al. Eponemycin exerts its antitumor effect through the inhibition of proteasome function. Cancer Res 1999; 59(12): 2798–801

  26. 26.

    Saghatelian A, Jessani N, Joseph A, et al. Activity-based probes for the proteomic profiling of metalloproteases. Proc Natl Acad Sci U S A 2004 Jul 6; 101: 10000–5

  27. 27.

    Kumar S, Zhou B, Liang F, et al. Activity-based probes for protein tyrosine phosphatases. Proc Natl Acad Sci U S A 2004 May 25; 101: 7943–8

  28. 28.

    Zhu Q, Huang X, Chen GYJ, et al. Activity based fluorescent probes that target phosphatases. Tetrahedron Lett 2003; 44: 2669–72

  29. 29.

    Adam GC, Sorensen EJ, Cravatt BF. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat Biotechnol 2002 Aug; 20: 805–9

  30. 30.

    Thornberry NA, Peterson EP, Zhao JJ, et al. Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 1994 Apr 5; (33): 3934–40

  31. 31.

    Hawthorne S, Hamilton R, Walker BJ, et al. Utilization of biotinylated diphenyl phosphonates for disclosure of serine proteases. Anal Biochem 2004 Mar 15; 326: 273–5

  32. 32.

    Zhu Q, Girish A, Chattopadhaya S, et al. Developing novel activity-based fluorescent probes that target different classes of proteases. Chem Commun (Camb) 2004 Jul; 7(13): 1512–3

  33. 33.

    Greenbaum DC, Arnold WD, Lu F, et al. Small molecule affinity fingerprinting: a tool for enzyme family subclassification, target identification, and inhibitor design. Chem Biol 2002 Oct; 9: 1085–94

  34. 34.

    Uhlmann F, Wernic D, Poupart MA, et al. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 2000 Oct 27; 103: 375–86

  35. 35.

    Salvesen GS. Caspases and apoptosis. Essays Biochem 2002; 38: 9–19

  36. 36.

    Lecaille F, Kaleta J, Bromme D. Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem Rev 2002 Dec; 102: 4459–88

  37. 37.

    Faleiro L, Kobayashi R, Fearnhead H, et al. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J 1997 May 1; 16: 2271–81

  38. 38.

    Mason RW, Wilcox D, Wikstrom P, et al. The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor. Biochem J 1989 Jan 1; 257: 125–9

  39. 39.

    Joyce JA, Baruch A, Chehade K, et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell 2004 May; 5: 443–53

  40. 40.

    Baruch A, Greenbaum D, Levy ET, et al. Defining a link between gap junction communication, proteolysis, and cataract formation. J Biol Chem 2001 Aug 3; 276: 28999–9006

  41. 41.

    Yasothornsrikul S, Greenbaum D, Medzihradszky KF, et al. CathepsinL in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter. Proc Natl Acad Sci USA 2003 Aug 5; 100: 9590–5

  42. 42.

    Greenbaum DC, Baruch A, Grainger M, et al. A role for the protease falcipain 1 in host cell invasion by the human malaria parasite. Science 2002 Dec 6; 298: 2002–6

  43. 43.

    Oleksy A, Golonka E, Banbula A, et al. Growth phase-dependent production of a cell wall-associated elastinolytic cysteine proteinase by Staphylococcus epidermidis. Biol Chem 2004 Jun; 385: 525–35

  44. 44.

    van der Hoorn RA, Leeuwenburgh MA, Bogyo M, et al. Activity profiling of papain-like cysteine proteases in plants. Plant Physiol 2004 Jul; 135: 1170–8

  45. 45.

    Boatright KM, Renatus M, Scott FL, et al. A unified model for apical caspase activation. Mol Cell 2003 Feb; 11: 529–41

  46. 46.

    Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001 Feb 15; 409: 860–921

  47. 47.

    Jessani N, Liu Y, Humphrey M, et al. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc Natl Acad Sci U S A 2002 Aug 6; 99: 10335–40

  48. 48.

    Overall CM, Lopez-Otin C. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2002 Sep; 2: 657–72

  49. 49.

    Puente XS, Sanchez LM, Overall CM, et al. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 2003 Jul; 4: 544–58

  50. 50.

    Chang C, Werb Z. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol 2001 Nov; 11: S37–43

  51. 51.

    Turner AJ, Isaac RE, Coates D. The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. Bioessays 2001 Mar; 23: 261–9

  52. 52.

    Garcia-Echeverria C, Traxler P, Evans DB. ATP site-directed competitive and irreversible inhibitors of protein kinases. Med Res Rev 2000 Jan; 20: 28–57

  53. 53.

    Colman RF. Affinity labeling of purine nucleotide sites in proteins. Annu Rev Biochem 1983; 52: 67–91

  54. 54.

    Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, et al. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 1996 Apr; 16: 1722–33

  55. 55.

    Mustelin T, Feng GS, Bottini N, et al. Protein tyrosine phosphatases. Front Biosci 2002 Jan 1; 7: d85–142

  56. 56.

    Ross DT, Scherf U, Eisen MB, et al. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 2000 Mar; 24: 227–35

  57. 57.

    Konecny G, Untch M, Pihan A, et al. Association of urokinase-type plasminogen activator and its inhibitor with disease progression and prognosis in ovarian cancer. Clin Cancer Res 2001 Jun; 7: 1743–9

  58. 58.

    Kanitakis J, Narvaez D, Claudy A. Differential expression of the CD10 antigen (neutral endopeptidase) in primary versus metastatic malignant melanomas of the skin. Melanoma Res 2002 Jun; 12: 241–4

  59. 59.

    Galardy RE, Grobelny D, Foellmer HG, et al. Inhibition of angiogenesis by the matrix metalloprotease inhibitor N-[R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]L-tryptophan methylamide. Cancer Res 1994 Sep 1; 54: 4715–8

  60. 60.

    Grobelny D, Poncz L, Galardy RE. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry 1992 Aug 11; 31: 7152–4

  61. 61.

    Solorzano CC, Ksontini R, Pruitt JH, et al. A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality. Shock 1997 Jun; 7: 427–31

  62. 62.

    Baruch A, Jeffery DA, Bogyo M. Enzyme activity: it’s all about image. Trends Cell Biol 2004 Jan; 14: 29–35

  63. 63.

    Gurtu V, Kain SR, Zhang G. Fluorometric and colorimetric detection of caspase activity associated with apoptosis. Anal Biochem 1997 Aug 15; 251: 98–102

  64. 64.

    Packard BZ, Toptygin DD, Komoriya A, et al. Design of profluorescent protease substrates guided by exciton theory. Methods Enzymol 1997; 278: 15–23

  65. 65.

    Komoriya A, Packard BZ, Brown MJ, et al. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J Exp Med 2000 Jun 5; 191: 1819–28

  66. 66.

    Hirata H, Takahashi A, Kobayashi S, et al. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J Exp Med 1998 Feb 16; 187: 587–600

  67. 67.

    Speers AE, Adam GC, Cravatt BF. Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne[3 + 2] cycloaddition. J Am Chem Soc 2003 Apr 23; 125: 4686–7

  68. 68.

    Speers AE, Cravatt BF. Profiling enzyme activities in vivo using click chemistry methods. Chem Biol 2004 Apr; 11: 535–46

  69. 69.

    Ovaa H, van Swieten PF, Kessler BM, et al. Chemistry in living cells: detection of active proteasomes by a two-step labeling strategy. Angew Chem Int Ed Engl 2003 Aug 11; 42: 3626–9

  70. 70.

    Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov 2002 Sep; 1: 727–30

  71. 71.

    Drews J. Drug discovery: a historical perspective. Science 2000 Mar 17; 287: 1960–4

  72. 72.

    Sordella R, Bell DW, Haber DA, et al. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004; 305: 1163–7

  73. 73.

    Leung D, Hardouin C, Boger DL, et al. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat Biotechnol 2003 Jun; 21: 687–91

Download references

Acknowledgments

This work was funded by a National Technology Centers for Networks and Pathways grant, NIH grant U54 RR020843 (awarded to Dr Bogyo). Dr Berger was funded by NHGRI trainiing grant 5T32HG00044.

Author information

Correspondence to Dr Matthew Bogyo.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Berger, A.B., Vitorino, P.M. & Bogyo, M. Activity-Based Protein Profiling. Am J Pharmacogenomics 4, 371–381 (2004). https://doi.org/10.2165/00129785-200404060-00004

Download citation

Keywords

  • Epidermal Growth Factor Receptor
  • Cysteine Protease
  • Fluorescence Resonance Energy Transfer
  • Fatty Acid Amide Hydrolase
  • Epidermal Growth Factor Receptor Gene