Abstract
The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ’s attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.
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Abbreviations
- CID:
-
collision-induced dissociation
- COFRADIC:
-
combined fractional diagonal chromatography
- CV:
-
coefficient of variation
- ETD:
-
electron transfer dissociation
- FDR:
-
false discovery rate
- HCD:
-
higher-energy collisional dissociation
- HILIC:
-
hydrophilic interaction liquid chromatography
- IMAC:
-
immobilized metal ion affinity chromatography
- IPTL:
-
isobaric peptide termini labelling
- iTRAQ:
-
isobaric tags for relative and absolute quantification
- NHS:
-
N-hydroxysuccinimide
- PQD:
-
pulsed Q dissociation
- PTM:
-
post-translational modification
- RP-HPLC:
-
reverse-phase high-performance liquid chromatography
- SCX:
-
strong cation exchange
- SILAC:
-
stable isotope labelling with amino acids in cell culture
- TMT:
-
tandem mass tag
- UHPLC:
-
ultra-high-performance liquid chromatography
- VSN:
-
variance-stabilizing normalization
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Acknowledgements
We thank the EPSRC for funding (EP/E053556/1) and the ChELSI initiative (EP/E036252/1). We thank Bruker Daltonik Bremen for help with the FT-ICR work (Drs Jens Fuchser and Matthias Witt).
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Published in the topical issue Quantitative Mass Spectrometry in Proteomics with guest editors Bernhard Kuster and Marcus Bantscheff.
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Evans, C., Noirel, J., Ow, S.Y. et al. An insight into iTRAQ: where do we stand now?. Anal Bioanal Chem 404, 1011–1027 (2012). https://doi.org/10.1007/s00216-012-5918-6
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DOI: https://doi.org/10.1007/s00216-012-5918-6