Skip to main content

iTRAQ-Based LC-LC MALDI TOF/TOF MS Quantitative Analysis of Membrane Proteins from Human Glioma

  • Protocol
  • First Online:
Neuroproteomics

Part of the book series: Neuromethods ((NM,volume 57))

  • 1984 Accesses

Abstract

Various proteomic approaches are being applied in brain tumor proteomics with regard to targeted proteins of interest, to discover phenotype specific markers which could facilitate diagnosis as well as potential antitumor drug targets. iTRAQ technology is a multiplexing protein quantitation strategy that provides relative and absolute measurements of protein abundance in complex mixtures based on the differential labeling of the proteins. Combined with membrane enrichment methodology, separation of the labeled peptides by two-dimensional liquid chromatography linked to tandem mass spectrometry and adequate data-mining, it can provide an excellent tool in search for novel isoform- and species-specific biomarkers and drug targets in various biotechnological and biomedical applications.Glioblastoma (GBM) is the most frequent primary brain tumor diagnosed in adults and remains one of the most lethal forms of human cancer. No biomarkers can distinguish different cell populations within GBMs or predict the potential of low grade gliomas to develop into malignant angiogenic gliomas. In our study, we have used iTRAQ-based technology to search for novel biomarkes of GBM.

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

References

  1. Rajcevic, U., Niclou, S. P. and Jimenez, C. R. (2009) Proteomics strategies for target identification and biomarker discovery in cancer Front Biosci 14, 3292–303.

    Article  PubMed  CAS  Google Scholar 

  2. Bantscheff, M., Schirle, M., Sweetman, G., Rick, J. and Kuster, B. (2007) Quantitative mass spectrometry in proteomics: a critical review Analytical and bioanalytical chemistry 389, 1017–31.

    Article  PubMed  CAS  Google Scholar 

  3. Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A. and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics Mol Cell Proteomics 1, 376–86.

    Article  CAS  Google Scholar 

  4. Kruger, M., Moser, M., Ussar, S., Thievessen, I., Luber, C. A., Forner, F., Schmidt, S., Zanivan, S., Fassler, R. and Mann, M. (2008) SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function Cell 134, 353–64.

    Article  PubMed  Google Scholar 

  5. Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J. and Mann, M. (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling Nat Biotechnol 21, 315–8.

    Article  PubMed  CAS  Google Scholar 

  6. Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M. and Klip, A. (2006) Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell ­culture (SILAC) J Proteome Res 5, 64–75.

    Article  PubMed  CAS  Google Scholar 

  7. Yao, X., Freas, A., Ramirez, J., Demirev, P. A. and Fenselau, C. (2001) Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus Anal Chem 73, 2836–42.

    Article  PubMed  CAS  Google Scholar 

  8. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H. and Aebersold, R. (1999) Quantitative analysis of complex protein ­mixtures using isotope-coded affinity tags Nat Biotechnol 17, 994–9.

    Article  PubMed  CAS  Google Scholar 

  9. Froment, C., Uttenweiler-Joseph, S., Bousquet-Dubouch, M. P., Matondo, M., Borges, J. P., Esmenjaud, C., Lacroix, C., Monsarrat, B. and Burlet-Schiltz, O. (2005) A quantitative proteomic approach using ­two-dimensional gel electrophoresis and ­isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity Proteomics 5, 2351–63.

    Article  PubMed  CAS  Google Scholar 

  10. Brand, M., Ranish, J. A., Kummer, N. T., Hamilton, J., Igarashi, K., Francastel, C., Chi, T. H., Crabtree, G. R., Aebersold, R. and Groudine, M. (2004) Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics Nat Struct Mol Biol 11, 73–80.

    Article  PubMed  CAS  Google Scholar 

  11. Ranish, J. A., Yi, E. C., Leslie, D. M., Purvine, S. O., Goodlett, D. R., Eng, J. and Aebersold, R. (2003) The study of macromolecular ­complexes by quantitative proteomics Nat Genet 33, 349–55.

    Article  PubMed  CAS  Google Scholar 

  12. Shiio, Y., Rose, D. W., Aur, R., Donohoe, S., Aebersold, R. and Eisenman, R. N. (2006) Identification and characterization of SAP25, a novel component of the mSin3 corepressor complex Mol Cell Biol 26, 1386–97.

    Article  PubMed  CAS  Google Scholar 

  13. Li, K. W., Hornshaw, M. P., Van Der Schors, R. C., Watson, R., Tate, S., Casetta, B., Jimenez, C. R., Gouwenberg, Y., Gundelfinger, E. D., Smalla, K. H. and Smit, A. B. (2004) Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of ­synaptic physiology The Journal of biological chemistry 279, 987–1002.

    CAS  Google Scholar 

  14. Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A. and Pappin, D. J. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics 3, 1154–69.

    Article  PubMed  CAS  Google Scholar 

  15. Keshamouni, V. G., Michailidis, G., Grasso, C. S., Anthwal, S., Strahler, J. R., Walker, A., Arenberg, D. A., Reddy, R. C., Akulapalli, S., Thannickal, V. J., Standiford, T. J., Andrews, P. C. and Omenn, G. S. (2006) Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype J Proteome Res 5, 1143–54.

    Article  PubMed  CAS  Google Scholar 

  16. de Souza, G. A., Godoy, L. M. and Mann, M. (2006) Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors Genome ­biology 7, R72.

    Article  PubMed  Google Scholar 

  17. Li, K. W., Miller, S., Klychnikov, O., Loos, M., Stahl-Zeng, J., Spijker, S., Mayford, M. and Smit, A. B. (2007) Quantitative proteomics and protein network analysis of hippocampal synapses of CaMKIIalpha mutant mice J Proteome Res 6, 3127–33.

    Article  PubMed  CAS  Google Scholar 

  18. Mukherjee, J., DeSouza, L. V., Micallef, J., Karim, Z., Croul, S., Siu, K. W. and Guha, A. (2009) Loss of collapsin response mediator Protein1, as detected by iTRAQ analysis, promotes invasion of human gliomas expressing mutant EGFRvIII Cancer Res 69, 8545–54.

    Article  PubMed  CAS  Google Scholar 

  19. Sakariassen, P. O., Prestegarden, L., Wang, J., Skaftnesmo, K. O., Mahesparan, R., Molthoff, C., Sminia, P., Sundlisaeter, E., Misra, A., Tysnes, B. B., Chekenya, M., Peters, H., Lende, G., Kalland, K. H., Oyan, A. M., Petersen, K., Jonassen, I., van der Kogel, A., Feuerstein, B. G., Terzis, A. J., Bjerkvig, R. and Enger, P. O. (2006) Angiogenesis-independent tumor growth mediated by stem-like cancer cells Proc Natl Acad Sci USA 103, 16466–71.

    Article  PubMed  CAS  Google Scholar 

  20. Hopkins, A. L. and Groom, C. R. (2003) Target analysis: a priori assessment of druggability Ernst Schering Res Found Workshop 11–7.

    Google Scholar 

  21. Josic, D. and Clifton, J. G. (2007) Mammalian plasma membrane proteomics Proteomics 7, 3010–29.

    Article  PubMed  CAS  Google Scholar 

  22. Josic, D., Clifton, J. G., Kovac, S. and Hixson, D. C. (2008) Membrane proteins as diagnostic biomarkers and targets for new therapies Curr Opin Mol Ther 10, 116–23.

    PubMed  CAS  Google Scholar 

  23. Rabilloud, T. (2003) Membrane proteins ride shotgun Nat Biotechnol 21, 508–10.

    Article  PubMed  CAS  Google Scholar 

  24. Rajcevic, U., Petersen, K., Knol, J. C., Loos, M., Bougnaud, S., Klychnikov, O., Li, K. W., Pham, T. V., Wang, J., Miletic, H., Peng, Z., Bjerkvig, R., Jimenez, C. R. and Niclou, S. P. (2009) iTRAQ based proteomic profiling reveals increased metabolic activity and cellular crosstalk in angiogenic compared to invasive Glioblastoma phenotype Mol Cell Proteomics.

    Google Scholar 

  25. Terzis, A. J., Niclou, S. P., Rajcevic, U., Danzeisen, C. and Bjerkvig, R. (2006) Cell therapies for glioblastoma Expert Opin Biol Ther 6, 739–49.

    Article  PubMed  CAS  Google Scholar 

  26. Wen, P. Y. and Kesari, S. (2008) Malignant gliomas in adults N Engl J Med 359, 492–507.

    Article  PubMed  CAS  Google Scholar 

  27. Carmeliet, P. and Jain, R. K. (2000) Angiogenesis in cancer and other diseases Nature 407, 249–57.

    Article  PubMed  CAS  Google Scholar 

  28. Jain, R. K., di Tomaso, E., Duda, D. G., Loeffler, J. S., Sorensen, A. G. and Batchelor, T. T. (2007) Angiogenesis in brain tumours Nat Rev Neurosci 8, 610–22.

    Article  PubMed  CAS  Google Scholar 

  29. Reiss, Y., Machein, M. R. and Plate, K. H. (2005) The role of angiopoietins during angiogenesis in gliomas Brain Pathol 15, 311–7.

    Article  PubMed  CAS  Google Scholar 

  30. Bogler, O. and Sawaya, R. (2008) Biomarkers and cancer stem cells in primary brain tumors. Foreword Curr Probl Cancer 32, 95–6.

    Article  PubMed  Google Scholar 

  31. Billecke, C., Malik, I., Movsisyan, A., Sulghani, S., Sharif, A., Mikkelsen, T., Farrell, N. P. and Bogler, O. (2006) Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles Mol Cell Proteomics 5, 35–42.

    PubMed  CAS  Google Scholar 

  32. Hamler, R. L., Zhu, K., Buchanan, N. S., Kreunin, P., Kachman, M. T., Miller, F. R. and Lubman, D. M. (2004) A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification Proteomics 4, 562–77.

    Article  PubMed  CAS  Google Scholar 

  33. Lubman, D. M., Kachman, M. T., Wang, H., Gong, S., Yan, F., Hamler, R. L., O’Neil, K. A., Zhu, K., Buchanan, N. S. and Barder, T. J. (2002) Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates J Chromatogr B Analyt Technol Biomed Life Sci 782, 183–96.

    Article  PubMed  CAS  Google Scholar 

  34. Wang, Y. C., Choi, M. H. and Han, J. (2004) Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves Anal Chem 76, 4426–31.

    Article  PubMed  CAS  Google Scholar 

  35. Furuta, M., Weil, R. J., Vortmeyer, A. O., Huang, S., Lei, J., Huang, T. N., Lee, Y. S., Bhowmick, D. A., Lubensky, I. A., Oldfield, E. H. and Zhuang, Z. (2004) Protein patterns and proteins that identify subtypes of glioblastoma multiforme Oncogene 23, 6806–14.

    Article  PubMed  CAS  Google Scholar 

  36. Guo, T., Wang, W., Rudnick, P. A., Song, T., Li, J., Zhuang, Z., Weil, R. J., DeVoe, D. L., Lee, C. S. and Balgley, B. M. (2007) Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens J Histochem Cytochem 55, 763–72.

    Article  PubMed  CAS  Google Scholar 

  37. Mustafa, D. A., Burgers, P. C., Dekker, L. J., Charif, H., Titulaer, M. K., Smitt, P. A., Luider, T. M. and Kros, J. M. (2007) Identification of glioma neovascularization-related proteins by using MALDI-FTMS and nano-LC fractionation to microdissected tumor vessels Mol Cell Proteomics 6, 1147–57.

    Article  PubMed  CAS  Google Scholar 

  38. Huang, P. H., Mukasa, A., Bonavia, R., Flynn, R. A., Brewer, Z. E., Cavenee, W. K., Furnari, F. B. and White, F. M. (2007) Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for ­glioblastoma Proc Natl Acad Sci USA 104, 12867–72.

    Article  PubMed  CAS  Google Scholar 

  39. Li, K.W, Hornshaw, M. P., van Minnen, J., Smalla, K. H., Gundelfinger, E. D. and Smit, A. B. (2005) Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass ­spectrometry to reveal post-synaptic density specific proteins J Proteome Res 4, 725–33.

    Google Scholar 

Download references

Acknowledgements

The work presented in this chapter was supported by EU FP6 Integrated project grant “Angiotargeting” (contract number 504743), by CRP-Santé, the Research Ministry (MCESR) in Luxembourg and by grant AFR-PDR-08-007 from the FNR, Luxembourg. The author is grateful to Dr. S.P. Niclou and Prof. R. Bjerkvig NorLux Neuro-Oncology laboratory, CRP-Santé, Luxembourg and Department of Biomedicine, University in Bergen, Norway for their invaluable support with this project and critical review of the manuscript. Drs. C. R. Jimenez and J. Knol, Oncoproteomics Laboratory, VU University Medical Center in Amsterdam, The Netherlands are kindly acknowledged for their help.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Uroš Rajčević .

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

Rajčević, U. (2011). iTRAQ-Based LC-LC MALDI TOF/TOF MS Quantitative Analysis of Membrane Proteins from Human Glioma. In: Li, K. (eds) Neuroproteomics. Neuromethods, vol 57. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-111-6_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-111-6_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-110-9

  • Online ISBN: 978-1-61779-111-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics