Protein Function Microarrays: Design, Use and Bioinformatic Analysis in Cancer Biomarker Discovery and Quantitation

  • Jessica Duarte
  • Jean-Michel Serufuri
  • Nicola Mulder
  • Jonathan BlackburnEmail author
Part of the Translational Bioinformatics book series (TRBIO, volume 3)


Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function, including in biomarker discovery applications. In this chapter, we review available methodologies relevant to this field and describe a simple approach to the design and fabrication of cancer-antigen arrays suitable for cancer biomarker discovery through serological analysis of cancer patients. We consider general issues that arise in antigen content generation, microarray fabrication and microarray-based assays and provide practical examples of experimental approaches that address these. We then focus on general issues that arise in raw data extraction, raw data preprocessing and analysis of the resultant preprocessed data to determine its biological significance, and we describe computational approaches to address these that enable quantitative assessment of serological protein microarray data. We exemplify this overall approach by reference to the creation of a multiplexed cancer-antigen microarray that contains 100 unique, purified, immobilised antigens in a spatially defined array, and we describe specific methods for serological assay and data analysis on such microarrays, including test cases with data originated from a malignant melanoma cohort.


Protein microarrays Cancer–testis antigens Cancer biomarker discovery Bioinformatic analysis Pipeline 



The authors thank Dr Aubrey Shoko, Dr Natasha Beeton-Kempen and Dr Judit Kumuthini for their help in generating the data herein. We thank the Centre for Proteomic & Genomic Research, Cape Town, for access to equipment and assistance in developing the CT100 array. JMB thanks the National Research Foundation (NRF), South Africa, for a Research Chair. The research was supported by grants from the NRF, University of Cape Town (UCT) and Marion Beatrice Waddel.


  1. Altman N. Replication, variation and normalization in microarray experiments. Appl Bioinformatics. 2005;4:1–23.CrossRefGoogle Scholar
  2. Anderson KS, LaBaer J. The sentinel within: exploiting the immune system for cancer biomarkers. J Proteome Res. 2005;4:1123–33.PubMedCrossRefGoogle Scholar
  3. Angenendt P, Glökler J, Sobek J, Lehrach H, Cahill DJ. Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications. J Chromatogr. 2003;1009:97–104.CrossRefGoogle Scholar
  4. Athappilly FK, Hendrickson WA. Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing. Structure. 1995;3:1407–19.PubMedCrossRefGoogle Scholar
  5. Beeton-Kempen N, Duarte JG, Shoko A, Safari Serufuri J-M, Cebon J, Blackburn JM. Monitoring melanoma patient responses to therapeutic vaccination using a cancer/testis antigen protein microarray. Manuscript submitted.Google Scholar
  6. Berrade L, Garcia AE, Camarero JA. Protein microarrays: novel developments and applications. Pharm Res. 2011;28:1480–99.PubMedCrossRefGoogle Scholar
  7. Blackburn JM, Shoko A. Protein function microarrays for customised systems-oriented proteomic analysis. In: Korf U, editor. Protein microarrays: methods and protocols, Methods in molecular biology. Springer protocols. New York: Humana Press; 2011. Chapter 21. ISBN 978-1-61779-285-4.CrossRefGoogle Scholar
  8. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–93.PubMedCrossRefGoogle Scholar
  9. Boutell JM, Hart DJ, Godber BLJ, Kozlowski RZ, Blackburn JM. Functional protein microarrays for parallel characterisation of p53 mutants. Proteomics. 2004;4:1950–8.PubMedCrossRefGoogle Scholar
  10. Boutros PC, Okey AB. Unsupervised pattern recognition: an introduction to the whys and wherefores of clustering microarray data. Brief Bioinform. 2005;6:331–43.PubMedCrossRefGoogle Scholar
  11. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.PubMedCrossRefGoogle Scholar
  12. Brusic V, Marina O, Wu CJ, Reinherz EL. Proteome informatics for cancer research: from molecules to clinic. Proteomics. 2007;7:976–91.PubMedCrossRefGoogle Scholar
  13. Büssow K, Konthur Z, Lueking A, Lehrach H, Walter G. Protein array technology: potential use in medical diagnostics. Am J Pharmacogenomics. 2001;1:1–7.CrossRefGoogle Scholar
  14. Casiano CA, Mediavilla-Varela M, Tan EM. Tumor-associated antigen arrays for the serological diagnosis of cancer. Mol Cell Proteomics. 2006;5:1745–59.PubMedCrossRefGoogle Scholar
  15. Causton HC, Quackenbush J, Brazma A. Microarray gene expression data analysis: a beginners guide. 1st ed. Malden: Blackwell Publishing; 2004.Google Scholar
  16. Chapman-Smith A, Cronan JE. The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity. Trends Biochem Sci. 1999;24:359–63.PubMedCrossRefGoogle Scholar
  17. Costello AB, Osborne JW. Best practices in exploratory factor analysis: four recommendations for getting the most from your analysis. Prac Assess Res Eval. 2005;10:1–9.Google Scholar
  18. Draghici S. Data analysis tools for DNA microarrays. 2nd ed. Boca Raton: Chapman & Hall; 2003.CrossRefGoogle Scholar
  19. Espina V, Mehta AI, Winters ME, Calvert V, Wulfkuhle J, Petricoin III EF, et al. Protein microarrays: molecular profiling technologies for clinical specimens. Proteomics. 2003;3:2091–100.PubMedCrossRefGoogle Scholar
  20. Fang Y, Lahiri J, Picard L. G protein-coupled receptor microarrays for drug discovery. Drug Discov Today. 2003;8:755–61.PubMedCrossRefGoogle Scholar
  21. Frank R, Hargreaves R. Clinical biomarkers in drug discovery and development. Nat Rev. 2003;2:566–80.CrossRefGoogle Scholar
  22. Freudenberg JM. Comparison of background correction and normalization procedures for high-density oligonucleotide microarrays. Leipzig Bioinformatics Working Paper. 2005;3:1–120.Google Scholar
  23. Gray MR, Colot HV, Guarente L, Rosbash M. Open reading frame cloning: identification, cloning, and expression of open reading frame DNA. Proc Natl Acad Sci U S A. 1982;79:6598–602.PubMedCrossRefGoogle Scholar
  24. Hall DA, Ptacek J, Snyder M. Protein microarray technology. Mech Ageing Dev. 2007;128:161–7.PubMedCrossRefGoogle Scholar
  25. Hardiman G. Microarray technologies – an overview. Pharmacogenomics. 2003;4:251–6.PubMedCrossRefGoogle Scholar
  26. Hastie T, Tibshirani R, Friedman J. The elements of statistical learning. 1st ed. New York: Springer; 2001.Google Scholar
  27. He M, Taussig MJ. Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method). Nucleic Acids Res. 2001;29:73–3.CrossRefGoogle Scholar
  28. Hultschig C, Kreutzberger J, Seitz H, Konthur Z, Bussow K, Lehrach H. Recent advances of protein microarrays. Curr Opin Chem Biol. 2006;10:4–10.PubMedCrossRefGoogle Scholar
  29. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 2009;37:211–15.CrossRefGoogle Scholar
  30. Ingvarsson J, Larsson A, Sjo AG, Truedsson L, Jansson B, Borrebaeck CAK, et al. Design of recombinant antibody microarrays for serum protein profiling: targeting of complement proteins research articles. J Proteome Res. 2007;6:3527–36.PubMedCrossRefGoogle Scholar
  31. Klein JB, Thongboonkerd V. Overview of proteomics. In: Thongboonkerd V, Klein JB, editors. Proteomics in nephrology. Basel: Karger; 2004. p. 1–10.Google Scholar
  32. Kodadek T. Protein microarrays: prospects and problems. Chem Biol. 2001;8:105–15.PubMedCrossRefGoogle Scholar
  33. Koopmann J-O, Blackburn J. High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays. Rapid Commun Mass Spectrom. 2003;17:455–62.PubMedCrossRefGoogle Scholar
  34. Lander ES, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921.PubMedCrossRefGoogle Scholar
  35. Lu T, Costello CM, Croucher PJP, Häsler R, Deuschl G, Schreiber S. Can Zipf’s law be adapted to normalize microarrays? BMC Bioinformatics. 2005;6:1–13.CrossRefGoogle Scholar
  36. Macbeath G. Protein microarrays and proteomics. Nat Genet. 2002;32:526–32.PubMedCrossRefGoogle Scholar
  37. MacBeath G, Schreiber SL. Printing proteins as microarrays for high-throughput function determination. Science. 2000;289:1760–3.PubMedGoogle Scholar
  38. Matarraz S, González-González M, Jara M, Orfao A, Fuentes M. New technologies in cancer. Protein microarrays for biomarker discovery. Clin Transl Oncol. 2011;13:156–61.PubMedCrossRefGoogle Scholar
  39. Michaud GA, Salcius M, Zhou F, Bangham R, Bonin J, Guo H, et al. Analyzing antibody specificity with whole proteome microarrays. Nat Biotechnol. 2003;21:1509–12.PubMedCrossRefGoogle Scholar
  40. Oshlack A, Emslie D, Corcoran LM, Smyth GK. Normalization of boutique two-color microarrays with a high proportion of differentially expressed probes. Genome Biol. 2007;8:2.1–8.CrossRefGoogle Scholar
  41. Phizicky E, Bastiaens PIH, Zhu H, Snyder M, Fields S. Protein analysis on a proteomic scale. Nature. 2003;422:208–15.PubMedCrossRefGoogle Scholar
  42. Ploner A, Miller LD, Hall P, Bergh J, Pawitan Y. Correlation test to assess low-level processing of high-density oligonucleotide microarray data. BMC Bioinformatics. 2005;6:1–20.CrossRefGoogle Scholar
  43. Predki PF. Functional protein microarrays: ripe for discovery. Curr Opin Chem Biol. 2004;8:8–13.PubMedCrossRefGoogle Scholar
  44. Quackenbush J. Computational analysis of microarray data. Genetics. 2001;2:418–27.PubMedGoogle Scholar
  45. Ramachandran N, Hainsworth E, Bhullar B, Eisenstein S, Rosen B, Lau AY, et al. Self-assembling protein microarrays. Science. 2004;305:86–90.PubMedCrossRefGoogle Scholar
  46. Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol. 2006;24:971–83.PubMedCrossRefGoogle Scholar
  47. Robinson WH. Antigen arrays for antibody profiling. Curr Opin Chem Biol. 2006;10:67–72.PubMedCrossRefGoogle Scholar
  48. Safari Serufuri J-M. Development of computational methods for Custom protein arrays analysis. A case study on a 100 protein (“ CT100 ”) cancer/testis antigen array. Masters thesis, University of Cape Town. 2010.Google Scholar
  49. Sambrook J, Russel DW, Macallum P. Molecular cloning – a laboratory manual. 3rd ed. Cold Spring Harbour: Cold Spring Harbour Laboratory Press; 2001.Google Scholar
  50. Sanchez-Carbayo M. Antibody arrays: technical considerations and clinical applications in cancer. Clin Chem. 2006;52:1651–9.PubMedCrossRefGoogle Scholar
  51. Scanlan MJ, Gure AO, Old LJ, Chen Y-t. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol Rev. 2002;188:22–32.PubMedCrossRefGoogle Scholar
  52. Schäferling M, Nagl S. Optical technologies for the read out and quality control of DNA and protein microarrays. Anal Bioanal Chem. 2006;385:500–17.PubMedCrossRefGoogle Scholar
  53. Schmidt DMZ, Mundorff EC, Dojka M, Bermudez E, Ness JE, Govindarajan S, et al. Evolutionary potential of (b/a)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily. Biochemistry. 2003;42:8387–93.PubMedCrossRefGoogle Scholar
  54. Schweitzer B, Predki P, Snyder M. Microarrays to characterize protein interactions on a whole-proteome scale. Proteomics. 2003;3:2190–9.PubMedCrossRefGoogle Scholar
  55. Smyth GK, Speed T. Normalization of cDNA microarray data. Methods. 2003;31:265–73.PubMedCrossRefGoogle Scholar
  56. Steinhoff C, Vingron M. Normalization and quantification of differential expression in gene expression microarrays. Brief Bioinform. 2006;7:166–77.PubMedCrossRefGoogle Scholar
  57. Tecan LSTM Series Laser Scanner: how to set the correct gain in the LS scanner.
  58. Tryfos P. Notes on Factor analysis. 2010.
  59. Ueda H, Howson JMM, Esposito L, Heward J, Snook H, Chamberlain G, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 2003;423:506–11.PubMedCrossRefGoogle Scholar
  60. Wikipedia. Factor analysis in psychometrics. 2010.
  61. Wilson DL, Buckley MJ, Helliwell CA, Wilson IW. New normalization methods for cDNA microarray data. Bioinformatics. 2003;19:1325–32.PubMedCrossRefGoogle Scholar
  62. Wise E, Yew WS, Babbitt PC, Gerlt JA, Rayment I. Homologous (b/a)8 -barrel enzymes that catalyze unrelated reactions: orotidine 5′-monophosphate decarboxylase and 3-keto-L-gulonate 6-phosphate decarboxylase. Biochemistry. 2002;41:3861–9.PubMedCrossRefGoogle Scholar
  63. Wolf-Yadlin A, Sevecka M, MacBeath G. Dissecting protein function and signaling using protein microarrays. Curr Opin Chem Biol. 2009;13:398–405.PubMedCrossRefGoogle Scholar
  64. Yang Y-S, Watson WJ, Tucker PW, Capra JD. Construction of recombinant DNA by exonuclease recession. Nucleic Acids Res. 1993;21:1889–93.PubMedCrossRefGoogle Scholar
  65. Zhao Y, Chapman DAG, Jones IM. Improving baculovirus recombination. Nucleic Acids Res. 2003;31:1–5.CrossRefGoogle Scholar
  66. Zhu H, Klemic JF, Chang S, Bertone P, Casamayor A, Klemic KG, et al. Analysis of yeast protein kinases using protein chips. Nat Genet. 2000;26:283–9.PubMedCrossRefGoogle Scholar
  67. Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, et al. Global analysis of protein activities using proteome chips. Science. 2001;14(293):2101–5.CrossRefGoogle Scholar
  68. Zhu X, Gerstein M, Snyder M. ProCAT: a data analysis approach for protein microarrays. Genome Biol. 2006;7:110.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jessica Duarte
    • 1
  • Jean-Michel Serufuri
    • 1
  • Nicola Mulder
    • 1
  • Jonathan Blackburn
    • 1
    Email author
  1. 1.Institute of Infectious Disease and Molecular Medicine, Faculty of Health SciencesUniversity of Cape TownCape TownSouth Africa

Personalised recommendations