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Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS


In this paper, we describe the use of iTRAQ (isobaric Tags for Relative and Absolute Quantitation) tags for comparison of protein expression levels between multiple samples. These tags label all peptides in a protein digest before labeled samples are pooled, fractionated and analyzed using mass spectrometry (MS). As the tags are isobaric, the intensity of each peak is the sum of the intensity of this peptide from all samples, providing a moderate enhancement in sensitivity. On peptide fragmentation, amino-acid sequence ions also show this summed intensity, providing a sensitivity enhancement. However, the distinct distribution of isotopes in the tags is such that, on further fragmentation, a tag-specific reporter ion is released. The relative intensities of these ions represent the relative amount of peptide in the analytes. Integration of the relative quantification data for the peptides allows relative quantification of the protein. This protocol discusses the rationale behind design, optimization and performance of experiments, comparing protein samples using iTRAQ chemistries combined with strong cation exchange chromatographic fractionation and MS.

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Figure 1: Expected data from a typical iTRAQ experiment.


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This work is funded in part by the Leukaemia and Lymphoma Research Fund, UK, and by the Manchester NIHR Biomedical Research Centre.

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Authors and Affiliations



R.D.U. and J.R.G. acquired the data for setup experiments; R.D.U. analyzed the data; R.D.U., J.R.G. and A.D.W. wrote the paper.

Corresponding author

Correspondence to Richard D Unwin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Example Excel workbook describing steps in global QC of internal control samples in an iTRAQ experiment. The first sheet, entitled 'Control data', contains protein identification data exported from ProteinPilot along with ratio data for replicate channels for those proteins selected using the parameters described in the text. In column K, these data are converted into log2 space. Below the list (row 1059) shows the plot of log2(Ratio) against unused protein score. From this table it can be seen that 95% of the proteins (1003) lie between log2(ratios) -0.35 and 0.35 (ratio 0.785 and 1.275), suggesting that, in a test vs control experiment, ratios outside these limits are likely to be due to biological, rather than technical, variation. The second sheet, entitled 'Histograms' shows a frequency histogram of these data generated by the Excel Histogram tool, with the frequency of ratios which occur in a given 'bin' plotted as both a line plot and bar chart to show that the control data are approximately normally distributed and centred around 0. (XLS 436 kb)

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Unwin, R., Griffiths, J. & Whetton, A. Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS. Nat Protoc 5, 1574–1582 (2010).

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