72 SPECTROSCOPY
ion is released in MS/MS spectrum it can be correlated to a particular sample source6
.
Two isobaric tag families are commercially available. Te first is the Tandem Mass Tag (TMT) reagent family which includes TMTzero, TMTduplex (reporting group mass 126 and 127 Da), TMTsixplex (reporting group mass 126 to 131 Da) and TMT10plex sets that are designed for a rapid and cost- effective evolution from method development to high-throughput protein quantitation.
Te mass reporter part is segregated from a mass normalisation moiety via a fragmentation vulnerable linker. Te TMTzero tag is suitable for testing and optimisation of sample preparation, fractionation and mass spectrometric fragmentation for peptide identification through reporter detection without using the more costly isotope-labelled tags1
.
three labelling methods discussed previously produce versions of each analysed peptide with mass variation at a particular amount. Te two forms will appear as two peaks with a certain mass difference in the mass spectrum.
Te mass-difference model is limited to a binary (2-plex), ternary (3-plex) or tertiary (4-plex) set of tags. Because of the limited plex numbers, comparison of multiple states cannot be achieved in one experiment. Tus, a multiplexed set of reagents for quantitative protein analysis has been developed.
Isobaric tagging is achieved by using chemical moieties or tags which are identical in mass to labelled peptides so that all derivatised peptides are isobaric, chromatographically indistinguishable and yielding a single peak in the mass spectrum for both samples (sick and non- sick samples).
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However, the relative abundances of the isobarically tagged peptides are revealed when the moieties fragment during MS/MS experiment to release reporter ions with different masses5
.
Te amine-reactive group of the tag (N-Hydroxysuccinimide ester- activated compounds) covalently binds to the peptide amino terminus and free amino termini of lysine residues of peptides and proteins with high efficiency.
In addition to the reactive group, the isobaric mass tags are designed to include two parts: a reporter or signature region and a mass normalising region. Te total mass of the reporter region and the normalising moieties is the same in all versions of the isobaric reagent, but the individual weights change. Consequently, when the reporter
Te other family is isobaric tags for absolute and relative quantification (iTRAQ), with up to 8-plex versions available with the following reporter ions/ isobaric tag values: 113/192, 114/191, 115/190, 116/189, 117/188, 118/187, 119/186, and 121/184 ensuring that all tags have a same total mass.
Conclusion Quantitative mass spectrometry protein analysis allows the comparison of protein expression between different states of biological systems. Te recent advances in this field combined with bioinformatics technologies, which are required to interpret complex data, are vital for biomarker discovery and drug target validation and will lead to clinical advantages for years to come.
Osama Chahrour and John Malone are with Almac Sciences, Craigavon, Northern Ireland, UK.
www.almacgroup.com
Fig. 2. (Left) ICAT reagents workflow.
REFERENCES: 1
Nakamura, T., and Oda, Y. (2007) Mass spectrometry- based quantitative proteomics. Biotechnol. Genet. Eng. Rev. 24,
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Ong, S.-E., and Mann, M. (2005)
Mass spectrometry-based proteomics turns quantitative.
Nat. Chem. Biol. 1, 252–62; 3
Petriz, B. A., Gomes, C. P., Rocha, L. A. O., Rezende, T. M. B., and Franco, O. L. (2012) Proteomics applied to exercise physiology: a cutting-edge technology. J. Cell.
Physiol. 227, 885–98; 4
Nesvizhskii, A. I., Vitek, O., and
Aebersold, R. (2007) Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat. Methods 4,
787–97.; 5
212–7; 6.
Domon, B., and Aebersold, R. (2006) Mass spectrometry and protein analysis. Science 312,
Gingras, A.-C., Gstaiger, M.,
Raught, B. & Aebersold, R. (2007) Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 8, 645–54.
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