Advertisement

Phosphopeptide Microarrays for Comparative Proteomic Profiling of Cellular Lysates

  • Liqian Gao
  • Hongyan Sun
  • Mahesh Uttamchandani
  • Shao Q. Yao
Part of the Methods in Molecular Biology book series (MIMB, volume 1002)

Abstract

Protein phosphorylation is one of the most important and well-studied posttranslational modifications. Aberrant phosphorylation causes a wide spectrum of diseases, including cancers. As a result, many of the proteins involved in these pathways are seen as vital drug targets and biomarkers in treatment and diagnosis. The availability of broad-based platforms that identify changes across cellular states is critical in understanding unique disease characteristics and changes at the proteomic level. To highlight how microarrays can be applied in this regard, we describe here a comparative proteomic profiling method using two-color sample labeling and application on phosphopeptide microarrays, followed by a pull-down strategy and MS-based protein identification. This strategy has been applied to uncover candidate biomarkers in breast cancer and colon cancer cell lines. Apart from the synthesis of the phosphopeptide libraries and growth/isolation of cellular lysates, the protocol takes approximately 15 days to complete, once key steps have been optimized, and can be readily extended to other similarly complex biological specimens/samples.

Key words

Phosphopeptide microarrays Biomarkers Protein phosphorylation Comparative proteomic profiling 

Notes

Acknowledgments

The authors acknowledge funding support from Ministry of Education (R-143-000-394-112), the Agency for Science, Technology and Research (R-143-000-391-305), and DSO National Laboratories.

References

  1. 1.
    Foong YM, Fu J, Yao SQ, Uttamchandani M (2012) Current advances in peptide and small molecule microarray technologies. Curr Opin Chem Biol 16:234–262PubMedCrossRefGoogle Scholar
  2. 2.
    Afshari CA, Nuwaysir EF, Barrett JC (1999) Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation. Cancer Res 59:4759–4760PubMedGoogle Scholar
  3. 3.
    Gao L, Uttamchandani M, Yao SQ (2012) Comparative proteomic profiling of mammalian cell lysates using phosphopeptide microarrays. Chem Commun 48:2240–2242CrossRefGoogle Scholar
  4. 4.
    Cohen P (2002) Protein kinases—the major drug targets of the twenty-first century? Nat Rev Drug Discov 1:309–315PubMedCrossRefGoogle Scholar
  5. 5.
    Johnson TO, Ermolieff J, Jirousek MR (2002) Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov 1:696–709PubMedCrossRefGoogle Scholar
  6. 6.
    Yaffe MB (2002) Phosphotyrosine-binding domains in signal transduction. Nat Rev Mol Cell Biol 3:177–186PubMedCrossRefGoogle Scholar
  7. 7.
    Sefton BM, Hunter T, Ball EH, Singer SJ (1981) Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell 24:165–174PubMedCrossRefGoogle Scholar
  8. 8.
    Hochgrafe F, Zhang L, O’Toole SA et al (2010) Tyrosine phosphorylation profiling reveals the signaling network characteristics of Basal breast cancer cells. Cancer Res 70:9391–9401PubMedCrossRefGoogle Scholar
  9. 9.
    Sabido E, Selevsek N, Aebersold R (2011) Mass spectrometry-based proteomics for systems biology. Curr Opin Biotechnol 23. doi: 10.1016/j.copbio.2011.11.014
  10. 10.
    Bodenmiller B, Aebersold R (2010) Quantitative analysis of protein phosphorylation on a system-wide scale by mass spectrometry-based proteomics. Methods Enzymol 470:317–334PubMedCrossRefGoogle Scholar
  11. 11.
    Arruda SC, Barbosa Hde S, Azevedo RA, Arruda MA (2011) Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics. Analyst 136:4119–4126PubMedCrossRefGoogle Scholar
  12. 12.
    Valledor L, Jorrin J (2011) Back to the basics: maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses. J Proteomics 74:1–18PubMedCrossRefGoogle Scholar
  13. 13.
    Kolch W, Pitt A (2010) Functional proteomics to dissect tyrosine kinase signalling pathways in cancer. Nat Rev Cancer 10:618–629PubMedCrossRefGoogle Scholar
  14. 14.
    Uttamchandani M, Lu CH, Yao SQ (2009) Next generation chemical proteomic tools for rapid enzyme profiling. Acc Chem Res 42:1183–1192PubMedCrossRefGoogle Scholar
  15. 15.
    Wu H, Ge J, Yang PY, Wang J, Uttamchandani M, Yao SQ (2011) A peptide aldehyde microarray for high-throughput profiling of cellular events. J Am Chem Soc 133:1946–1954PubMedCrossRefGoogle Scholar
  16. 16.
    Shi H, Uttamchandani M, Yao SQ (2011) Applying small molecule microarrays and resulting affinity probe cocktails for proteome profiling of mammalian cell lysates. Chem Asian J 6:2803–2815PubMedCrossRefGoogle Scholar
  17. 17.
    Uttamchandani M, Lee WL, Wang J, Yao SQ (2007) Quantitative inhibitor fingerprinting of metalloproteases using small molecule microarrays. J Am Chem Soc 129:13110–13117PubMedCrossRefGoogle Scholar
  18. 18.
    Kattah MG, Alemi GR, Thibault DL, Balboni I, Utz PJ (2006) A new two-color Fab labeling method for autoantigen protein microarrays. Nat Methods 3:745–751PubMedCrossRefGoogle Scholar
  19. 19.
    Lee WL, Li J, Uttamchandani M, Sun H, Yao SQ (2007) Inhibitor fingerprinting of metalloproteases using microplate and microarray platforms: an enabling technology in Catalomics. Nat Protoc 2:2126–2138PubMedCrossRefGoogle Scholar
  20. 20.
    Sun H, Lu CH, Shi H, Gao L, Yao SQ (2008) Peptide microarrays for high-throughput studies of Ser/Thr phosphatases. Nat Protoc 3:1485–1493PubMedCrossRefGoogle Scholar
  21. 21.
    Chattopadhaya S, Tan LP, Yao SQ (2006) Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays. Nat Protoc 1:2386–2398PubMedCrossRefGoogle Scholar
  22. 22.
    Tannu NS, Hemby SE (2006) Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling. Nat Protoc 1:1732–1742PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Liqian Gao
    • 1
  • Hongyan Sun
    • 2
  • Mahesh Uttamchandani
    • 3
  • Shao Q. Yao
    • 1
  1. 1.National University of SingaporeSingaporeRepublic of Singapore
  2. 2.City University of Hong KongHong KongChina
  3. 3.DSO National LaboratoriesSingaporeRepublic of Singapore

Personalised recommendations