Skip to main content

Differential Proteomics Incorporating iTRAQ Labeling and Multi-dimensional Separations

  • Protocol
  • First Online:
Drug Safety Evaluation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 691))

Abstract

Considerable effort is currently being expended to integrate newly developed “omics”-based approaches (proteomics, transcriptomics, and metabonomics) into preclinical safety evaluation workflows in the hope that more sensitive prediction of toxicology can be achieved as reported by Waters and Fostel (Nat. Rev. Genet. 5(12):936–948, 2004) and Craig et al. (J. Proteome Res. 5(7):1586–1601, 2006). Proteomic approaches are well placed to contribute to this effort as (a) proteins are the metabolically active products of genes and, as such, may provide more sensitive and direct predictive information on drug-induced liabilities and (b) they have the potential to determine tissue leakage markers in peripheral fluids. Here, we describe a workflow for proteomic semi-quantitative expression profiling of liver from rats treated with a known hepatotoxicant using a multiplexed isobaric labeling strategy and multi-dimensional liquid chromatography.

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

Access this chapter

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 159.00
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

Institutional subscriptions

Similar content being viewed by others

References

  1. Collins, B.C., et al. (2007) Use of proteomics for the discovery of early markers of drug ­toxicity. Expert Opin. Drug Metab. Toxicol. 3(5), 689–704.

    Article  CAS  PubMed  Google Scholar 

  2. Merrick, B.A. (2006) Toxicoproteomics in liver injury and inflammation. Ann. N. Y. Acad. Sci. 1076, 707–717.

    Article  CAS  PubMed  Google Scholar 

  3. Merrick, B.A. (2008) The plasma proteome, adductome and idiosyncratic toxicity in toxicoproteomics research. Brief Funct. Genomic Proteomic 7(1), 35–49.

    Article  CAS  PubMed  Google Scholar 

  4. Wetmore, B.A., and Merrick, B.A. (2004) Toxicoproteomics: proteomics applied to toxicology and pathology. Toxicol. Pathol. 32(6), 619–642.

    Article  CAS  PubMed  Google Scholar 

  5. Aggarwal, K., Choe, L.H., and Lee, K.H. (2006) Shotgun proteomics using the iTRAQ isobaric tags. Brief Funct. Genomic Proteomic 5(2), 112–120.

    Article  CAS  PubMed  Google Scholar 

  6. Fella, K., et al. (2005) Use of two-dimensional gel electrophoresis in predictive toxicology: identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis. Proteomics 5(7), 1914–1927.

    Article  CAS  PubMed  Google Scholar 

  7. Kersey, P.J., et al. (2004) The International Protein Index: an integrated database for ­proteomics experiments. Proteomics 4(7), 1985–1988.

    Article  CAS  PubMed  Google Scholar 

  8. iTRAQ Reagents Chemistry Reference Guide. (2004) [http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_041463.pdf]

  9. Essader, A.S., et al. (2005) A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5(1), 24–34.

    Article  CAS  PubMed  Google Scholar 

  10. Lengqvist, J., Uhlen, K., and Lehtio, J. (2007) iTRAQ compatibility of peptide immobilized pH gradient isoelectric focusing. Proteomics 7(11), 1746–1752.

    Article  CAS  PubMed  Google Scholar 

  11. Elias, J.E., and Gygi, S.P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4(3), 207–214.

    Article  CAS  PubMed  Google Scholar 

  12. Martens, L., Vandekerckhove, J., and Gevaert, K. (2005) DBToolkit: processing protein databases for peptide-centric proteomics. Bio­informatics 21(17), 3584–3585.

    Article  CAS  PubMed  Google Scholar 

  13. Yang, Y., et al. (2007) A comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-based protein identification and iTRAQ-based shotgun quantitative proteomics. J. Biomol. Tech. 18(4), 226–237.

    PubMed  Google Scholar 

  14. Griffin, T.J., et al. (2007) iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J. Proteome Res. 6(11), 4200–4209.

    Article  CAS  PubMed  Google Scholar 

  15. Keller, A., et al. (2005) A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol. Syst. Biol. Article number: 1, 2005.0017. pages 1–8.

    Google Scholar 

Download references

Acknowledgments

We would like to thank all the members of the PredTox Consortium. Funding is acknowledged under the EU FP6 Integrated Project, InnoMed. The UCD Conway Institute and the Proteome Research Centre is funded by the Programme for Research in Third Level Institutions (PRTLI), as administered by the Higher Education Authority (HEA) of Ireland.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to William M. Gallagher .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Collins, B.C., Lau, T.Y.K., Pennington, S.R., Gallagher, W.M. (2011). Differential Proteomics Incorporating iTRAQ Labeling and Multi-dimensional Separations. In: Gautier, JC. (eds) Drug Safety Evaluation. Methods in Molecular Biology, vol 691. Humana Press. https://doi.org/10.1007/978-1-60761-849-2_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-849-2_23

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-186-8

  • Online ISBN: 978-1-60761-849-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics