Skip to main content
SpringerLink
Log in
Book cover

Chemical Genomics and Proteomics pp 133–162Cite as

Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 800))

Abstract

Systematic analysis of protein and enzyme function typically requires scale-up of protein expression and purification prior to assay development; this can often be limiting. Miniaturization of assays provides an alternative approach, but simple, generic methods are in short supply. Here we show how custom microarrays can be adapted to this purpose. We discuss the different routes to array fabrication and describe in detail one facile approach in which the purification and immobilization procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of arrays of human protein kinases and of human cytochrome P450s. We discuss methods for both ligand-binding and turnover-based assays, as well as data analysis on such arrays.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hunter S, Apweiler R et al. (2009) InterPro: the integrative protein signature database (2009). Nucleic Acids Res. 37: D224–228

    Article  Google Scholar 

  2. Wise EY, Yew WS, Babbitt PC, Gerlt JA, Rayment I (2002) Homologous (b/a)8-Barrel Enzymes That Catalyze Unrelated Reactions: Orotidine 5’-Monophosphate Decarboxylase and 3-Keto-L-Gulonate 6-Phosphate Decar-boxylase. Biochemistry 41: 3861–3869

    Article  PubMed  CAS  Google Scholar 

  3. Schmidt, DMZ, Mundorff EC, Dojka M, Bermudez E et al (2003) Evolutionary potential of (b/a)8-Barrels: Functional promiscuity produced by single substitutions in the enolase superfamily. Biochemistry 42: 8387–8393

    Article  PubMed  CAS  Google Scholar 

  4. The genome international sequencing consortium (2001) Initial sequencing and analysis of the human genome. Nature 409: 860–921

    Article  Google Scholar 

  5. MacBeath, G (2002) Protein microarrays and proteomics. Nature Genetics 32: 526–532

    Article  PubMed  CAS  Google Scholar 

  6. Wolf-Yadlin A, Sevecka M, MacBeath G (2009) Dissecting protein function and signaling using protein microarrays. Current Op Chem Biol 13: 398–405

    Article  CAS  Google Scholar 

  7. Boutell JM, Hart DJ, Godber BLJ, Kozlowski RZ, Blackburn JM. (2004) Analysis of the effect of clinically-relevant mutations on p53 function using protein microarray technology. Proteomics 4: 1950–1958

    Article  PubMed  CAS  Google Scholar 

  8. Kodadek T (2001) Protein microarrays: prospects and problems. Chem Biol 8:105–115

    Article  PubMed  CAS  Google Scholar 

  9. Predki P (2004) Functional protein microarrays: Ripe for discovery. Curr Opin Chem Biol 8: 8–13

    Article  PubMed  CAS  Google Scholar 

  10. Zhu H, Klemic JF, Chang S, Bertone P et al. (2000) Analysis of yeast protein kinases using protein chips. Nat Genet 26: 283–289

    Article  PubMed  CAS  Google Scholar 

  11. Zhu H, Bilgin M, Bangham R, Hall D et al. (2001) Global analysis of protein activities using proteome chips. Science 293  : 2101–2105

    Article  PubMed  CAS  Google Scholar 

  12. Michaud GA, Salcius M, Zhou F, Bangham R. et al. (2003) Analyzing antibody specificity with whole proteome microarrays. Nature Biotechnology 21: 1509–12

    Article  PubMed  CAS  Google Scholar 

  13. Fang Y, Lahiri J, Picard L (2003) G-Protein-coupled receptor microarrays for drug discovery. Drug Discovery Today 8: 755–761

    Article  PubMed  CAS  Google Scholar 

  14. MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289: 1760–1763

    PubMed  CAS  Google Scholar 

  15. Angenendt P, Glokler J, Sobek J, Lehrach H, Cahill DJ (2003) Next generation of protein microarray support materials: Evaluation for protein and antibody microarray applications. J Chromatogr A 1009: 97–104

    Article  PubMed  CAS  Google Scholar 

  16. Koopmann J-O, Blackburn JM (2003) High Affinity Capture Surface for MALDI compatible Protein Microarrays. Rapid Communication in Mass Spectrometry 17: 1–8

    Article  Google Scholar 

  17. He M, Taussig M (2001) Single step generation of protein arrays from DNA by cell-free expression and in situ immobilization (PISA method). Nucleic Acids Res 29: E73.

    Article  PubMed  CAS  Google Scholar 

  18. Ramachandran N, Hainsworth E et al. (2004) Self-Assembling Protein Microarrays. Science 305: 86–90

    Article  PubMed  CAS  Google Scholar 

  19. Zhao Y, Chapman DAG, Jones IM (2003) Improving baculovirus recombination. Nucleic Acids Res.31: e6

    Article  PubMed  Google Scholar 

  20. Sambrook J, MacCallum P, Russell D (2001) Molecular Cloning, A Laboratory Manual, Third ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  21. Athappilly FK, Hendrickson WA (1995) Structure of the biotinyl domain of acetyl-coenzymeA carboxylase determined by MAD phasing. Structure 3: 1407–19

    Article  PubMed  CAS  Google Scholar 

  22. Chapman-Smith A, Cronan JE (1999) The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity. Trends Biochem Sci 24: 359–363

    Article  PubMed  CAS  Google Scholar 

  23. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714–2723

    Article  PubMed  CAS  Google Scholar 

  24. Yang Y-S, Watson WJ, Tucker PW, Capra JD (1993) Construction of recombinant DNA by exonuclease recession. Nucleic Acids Res 21: 1889–1893

    Article  PubMed  CAS  Google Scholar 

  25. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254

    Article  PubMed  CAS  Google Scholar 

  26. Terwilliger TC, Stuart D, Yokoyama S (2009) Lessons from Structural Genomics. Ann Rev Biophys 38: 371–383

    Article  CAS  Google Scholar 

  27. Brown M (2007) Novel fluorescent kinase ligands and assays employing the same. Patent application no. WO2008071937

    Google Scholar 

  28. Hernandez-Boussard T, Whirl-Carrillo M, Hebert J.M, et al (2008) The pharmacogenetics and pharmacogenomics database: accentuating the knowledge. Nucleic Acids Research 36 (Database issue), D913–D918

    Google Scholar 

  29. Ingelman-Sundberg M (2004) Pharmacogen-etics of cytochrome P450 and its applications in drug therapy: the past, present and future. TRENDS in Pharmacological Sciences 25: 193–200

    Article  PubMed  CAS  Google Scholar 

  30. Choi-Rhee E, Cronan JE (2003) The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 278: 30806–30812

    Article  PubMed  CAS  Google Scholar 

  31. Cull MG, Schatz PJ (2000) Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol 326: 430–40

    Article  PubMed  CAS  Google Scholar 

  32. Lue RY, Chen GY, Hu Y, Zhu Q, Yao SQ (2004) Versatile protein biotinylation strategies for potential high-throughput proteomics. J Am Chem Soc 126: 1055–62

    Article  PubMed  CAS  Google Scholar 

  33. Berliner E, Mahtani HK, Karki S, Chu LF, et al. (1994) Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. J Biol Chem 269: 8610–5

    PubMed  CAS  Google Scholar 

  34. Lerner CG, Saiki AY (1996) Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells. Anal. Biochem 240: 185–96

    Article  PubMed  CAS  Google Scholar 

  35. Parrott MB, Barry MA (2001) Metabolic biotinylation of secreted and cell surface proteins from mammalian cells. Biochem Biophys Res Commun 281: 993–1000

    Article  PubMed  CAS  Google Scholar 

  36. Zheng J, Li L, et al (2005) Strong Repulsive Forces between Protein and Oligo (ethylene glycol) Self- Assembled Monolayers: A Molecular Simulation Study. Biophys J. 89: 158–166

    Article  PubMed  CAS  Google Scholar 

  37. Makris TM, von Koenig K, Schlichting I, Sligar, SG (2006) The status of high-valent metal oxo complexes in the P450 cytochromes. Journal of Inorganic Biochemistry 100: 507–518

    Article  PubMed  CAS  Google Scholar 

  38. Pylypenko O, Schlichting I (2004) Structural aspects of ligand binding to and electron transfer in bacterial and fungal P450s. Annual Reviews in Biochemistry 73: 991–1018

    Article  CAS  Google Scholar 

  39. Sakai-Kato K, Kato M, Homma H, Toyo’oko T, Utsunomiya-Tate N (2005) Creation of a P450 array toward high-throughput analysis. Analytical Chemistry 77: 7080–7083

    Article  PubMed  CAS  Google Scholar 

  40. Lee M, Park CB, Dordick JS, Clark DS (2005) Metabolising enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proceedings of the National Academy of Sciences USA 102: 983–987

    Article  CAS  Google Scholar 

  41. Sukumaran SM, Potsaid B, Lee M, Clark DS, Dordick JS (2009) Development of a fluorescence-based, ultra high-throughput screening platform for nanoliter-scale cytochrome P450 microarrays. Journal of Biomolecular Screening 14: 668–678

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Nashied Peton, Sarah Joyce, Colin Wheeler, Jens Koopman, Nick Workman, Steve Parham, and Mike McAndrew for their help in generating the data detailed herein. We thank Procognia Ltd (UK) for provision of human kinase arrays and also thank the Centre for Proteomic & Genomic Research, Cape Town, for access to equipment. JMB thanks the National Research Foundation (NRF; South Africa) for a Research Chair; NBK thanks the NRF for a PhD studentship. The research was supported by grants from the NRF, Procognia Ltd and Genetix PLC (UK).

Author information

Authors and Affiliations

  1. Division of Medical Biochemistry, Institute for Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

    Jonathan M. Blackburn & Natasha Beeton-Kempen

  2. Centre for Proteomic and Genomic Research, Cape Town, South Africa

    Aubrey Shoko

Authors
  1. Jonathan M. Blackburn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aubrey Shoko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Natasha Beeton-Kempen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jonathan M. Blackburn .

Editor information

Editors and Affiliations

  1. Royston, SG8 0ZN, Hertfordshire, United Kingdom

    Edward D. Zanders

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Blackburn, J.M., Shoko, A., Beeton-Kempen, N. (2012). Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function. In: Zanders, E. (eds) Chemical Genomics and Proteomics. Methods in Molecular Biology, vol 800. Humana Press. https://doi.org/10.1007/978-1-61779-349-3_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-349-3_10

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-348-6

  • Online ISBN: 978-1-61779-349-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation