Quantification of Proteins by iTRAQ - LC-MS/MS in Proteomics: Methods and Applications - page 204

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B
Fig. 12.1 A generic iTRAQ experiment. ( A ) A schematic of two iTRAQ tags, showing three distinct groups, a peptide-
reactive group, the reporter group, which allows relative quantification upon MS/MS, and a balance group, which main-
tains the isobaric nature of the tags. ( B ) An overview of an iTRAQ workflow. Peptides from multiple samples are labelled
with iTRAQ tags. These samples are pooled, generating a single ion for each peptide. One putative peptide ion (marked
with a ∗ in the figure) is then selected and fragmented. A typical MS/MS spectrum is shown, demonstrating sequence
identification, with the reporter ion region expanded to demonstrate how peptide-relative quantification is determined.
analysed simultaneously, the same peptide from the same samples
will appear at the same mass in MS. However, upon fragmen-
tation of the peptides by collision-induced dissociation (CID),
the peptide fragments provide amino acid sequence information
(and therefore peptide identity) and the iTRAQ tag fragments to
release a tag-specific reporter ions. The ratios of these reporter
ions are representative of the proportions of that peptide in each
sample ( Fig. 12.1B ) .
This principle was first demonstrated by Thompson et al.
( 1 ) who synthesised peptides containing a 'tandem mass tag'
and showed that this strategy could indeed be used to obtain
relative quantification in tandem MS experiment. A year later,
Ross et al. published a similar approach using iTRAQ (iso-
baric tags for relative and absolute quantification) ( 2 ) , where
they described a tag which contained a reactive moiety-enabling
reaction with any peptide (a feature postulated but not shown
by Thompson et al.). The remainder of this chapter will
deal exclusively with the iTRAQ tagging technology, available
through AB Sciex as either a four-channel or eight-channel