Quantification of Proteins by iTRAQ

  • Richard D. Unwin
Part of the Methods in Molecular Biology book series (MIMB, volume 658)


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.

Key words

Peptide protein iTRAQ isobaric relative quantitation liquid chromatography mass spectrometry 



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.


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Richard D. Unwin
    • 1
  1. 1.Stem Cell and Leukaemia Proteomics LaboratoryUniversity of ManchesterManchesterUK

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