Abstract
Protein relative quantification is a key facet of many proteomics experiments. Several methods exist for this type of work, some of which are described elsewhere in this volume. In this chapter we will describe the use of isobaric tags for relative and absolute quantification (iTRAQ). These chemical tags attach to all peptides in a protein digest via free amines at the peptide N-terminus and on the side chain of lysine residues. Labelled samples are then pooled and analysed simultaneously. Since the tags are isobaric, labelled peptides do not show a mass shift in MS, instead signal from the same peptide from all samples is summed, providing a moderate increase in sensitivity. Upon peptide fragmentation, sequence ions (b- and y-type) also show this summed intensity which aids sensitivity. However, the distribution of isotopes in the different tags is such that when the tags fragment a tag-specific ‘reporter’ ion is released. The ratio of signal intensities from these tags acts as an indication of the relative proportions of that peptide between the different labelled samples. This chapter will describe the procedure for labelling and analysing peptide/protein samples using iTRAQ.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Thompson, A., Schafer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., Neumann, T., and Hamon, C. (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904.
Ross, P. L., Huang, Y. L. 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, 1154–1169.
Ogata, Y., Charlesworth, M. C., Higgins, L., Keegan, B. M., Vernino, S., and Muddiman, D. C. (2007) Differential protein expression in male and female human lumbar cerebrospinal fluid using iTRAQ reagents after abundant protein depletion. Proteomics 7, 3726–3734.
Hardt, M., Witkowska, H. E., Webb, S., Thomas, L. R., Dixon, S. E., Hall, S. C. and Fisher, S. J. (2005) Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents. Anal. Chem. 77, 4947–4954.
Kristiansson, M. H., Bhat, V. B., Babu, I. R., Wishnok, J. S., and Tannenbaum, S. R. (2007) Comparative time-dependent analysis of potential inflammation biomarkers in lymphoma-bearing SJL mice. J. Proteome Res. 6, 1735–1744.
DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J. Z., Romaschin, A. D., Colgan, T. J. and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and clCAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377–386.
Bouchal, P., Roumeliotis, T., Hrstka, R., Nenutil, R., Vojtesek, B. and Garbis, S. D. (2009) Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis. J. Proteome Res. 8, 362–373.
Garbis, S. D., Tyritzis, S. I., Roumeliotis, T., Zerefos, P., Giannopoulou, E. G., Vlahou, A., Kossida, S., Diaz, J., Vourekas, S., Tamvakopoulos, C., Pavlakis, K., Sanoudou, D., and Constantinides, C. A. (2008) Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 7, 3146–3158.
Unwin, R. D., Smith, D. L., Blinco, D., Wilson, C. L., Miller, C. J., Evans, C. A., Jaworska, E., Baldwin, S. A., Barnes, K., Pierce, A., Spooncer, E., and Whetton, A. D. (2006) Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells. Blood 107, 4687–4694.
Schnölzer, M., Jedrzejewski, P., and Lehmann, W. D. (1996) Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17, 945–953.
Faca, V., Coram, M., Phanstiel, D., Glukhova, V., Zhang, Q., Fitzgibbon, M., McIntosh, M., and Hanash, S. (2006) Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS. J. Proteome Res. 5, 2009–2018.
Williamson, A. J. K., Smith, D. L., Blinco, D., Unwin, R. D., Pearson, S., Wilson, C., Miller, C., Lancashire, L., Lacaud, G., Kouskoff, V., and Whetton, A. D. (2008) Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis. Mol. Cell Proteomics 7, 459–472.
Pierce, A., Unwin, R. D., Evans, C. A., Griffiths, S., Carney, L., Zhang, L., Jaworska, E., Lee, C. F., Blinco, D., Okoniewski, M. J., Miller, C. J., Bitton, D. A., Spooncer, E., and Whetton, A. D. (2008) Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases. Mol. Cell Proteomics 7, 853–863.
Trinidad, J. (2007) Quantitative analysis of synaptic phosphorylation and protein expression. Mol. Cell Proteomics 7, 684–696.
Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D. A., and White, F. M. (2007) Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc. Natl. Acad. Sci. USA 104, 5860–5865.
Zhang, Y., Wolf-Yadlin, A., Ross, P. L., Pappin, D. J., Rush, J., Lauffenburger, D. A., and White, F. M. (2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol. Cell Proteomics 4, 1240–1250.
Wiese, S., Reidegeld, K. A., Meyer, H. E., and Warscheid, B. (2007) Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics, 7, 340–350.
Shilov, I. V., Seymour, S. L., Patel, A. A., Loboda, A., Tang, W. H., Keating, S. P., Hunter, C. L., Nuwaysir, L. M., and Schaeffer, D. A. (2007) The paragon algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol. Cell Proteomics 6, 1638–1655.
Acknowledgements
The author would like to thank Prof. Tony Whetton, University of Manchester, for encouragement and advice. This work is partially funded by Leukaemia Research Fund, UK and the NIHR Manchester Biomedical Research Centre.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Unwin, R.D. (2010). Quantification of Proteins by iTRAQ. In: Cutillas, P., Timms, J. (eds) LC-MS/MS in Proteomics. Methods in Molecular Biology, vol 658. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-780-8_12
Download citation
DOI: https://doi.org/10.1007/978-1-60761-780-8_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-779-2
Online ISBN: 978-1-60761-780-8
eBook Packages: Springer Protocols