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iTRAQ gets put to the test
experimental rigor to an anecdotal numbers with a great degree of cauproblem. tion,” Wright says. “The direction [of Proteomics used to be an exercise in “I think [the study] is elegantly put change] is fine, but you need to be protein cataloging. Today, it is mostly together,” he says. “Although people very aware of where these numbers about quantitation, and isobaric tags recognize this as a potential problem come from and what the caveats on such as iTRAQ from Life Technologies, with iTRAQ, at the same time, often their values mean.” Inc., and TMT from Thermo Fisher Scipeople don’t set up studies designed to Aaron Hudson, senior manager of entific, Inc., have become popular tease this phenomenon apart.” the proteomics marketing team at Life tools. In particular, Marto says the characTechnologies, says the company has Now that these reagents are in wideterization of the phenylalanine immoaddressed these issues with more sospread use, however, some researchers nium ion contamination of the question their accuracy. Specif121 m/z iTRAQ channel “will ically, anecdotal evidence sugraise awareness” of this previgests the reagents tend to unously overlooked potential derestimate protein source of error. But, he adds, abundance. In a recent JPR additional work is required to paper (2009, DOI 10.1021/ tease out some of the nuances pr900634c), Phillip Wright of of iTRAQ data analysis. “The the University of Sheffield last couple of recommenda(U.K.) and colleagues decided tions highlight the real need for to settle the matter once and a true error model in terms of for all. evaluating the statistical signifiUsing calibrated protein cance of the [reporter] ratios,” standards mixed in precise rahe says. tios, with or without a complex Hanno Steen, an iTRAQ and peptide background matrix, TMT user at Children’s HospiWright and his team measured Off-TRAQ. Phenylalanine-derived [M+1] immonium ions (m/z tal Boston, calls the study how closely iTRAQ quantita121.08) can be resolved from the iTRAQ m/z 121 reporter ions “very, very useful,” adding, “It tion matched the known abun(m/z 121.12) in (a) high-resolution MS/MS spectra but not at (b) would be great if these guidedances in each sample and low resolution. lines were made publicly availhow tightly replicate values able. Applied Biosystems clustered to one another. They should put these guidelines dub their verdict “the good, into their manual.” phisticated algorithms for iTRAQ quanthe bad, and the ugly” of iTRAQ Steen says the paper will also cause titation in its newest ProteinPilot softquantitation. him to scrutinize his own data more ware releasesassuming those data are “The good is the potential for iTRAQ closely. “I will definitely look much collected using Applied Biosystems to provide accurate quantification spanmore into the phenylalanine issue, and mass spectrometers, that is. ning two orders of magnitude,” the auwill make sure all my postdocs read “If you are using the reagents acthors write. However, that potential is this paper so that when they do their cording to the instructions in the kit limited by two factors: isotopic impuridata analysis, they keep all these issues and with our latest version of software, ties (“the bad”), a manufacturing issue; in mind,” he says. then the issues Phil raises are diminand peptide cofragmentation (“the ugly”; As for Wright, he continues to use ished,” Hudson says. inadvertently selecting two or more iTRAQ, though not exclusively. In a For those requiring more precise closely spaced peptides for MS/MS inproperly designed experiment, users quantitation, Hudson recommends apstead of one), a problem of sample comcan safely rely on iTRAQ-reported plying Life Technologies’ related plexity. The former can easily be cortrends, he says, but not their magnimTRAQ reagents to targeted multiplerected for using manufacturer values, the tudes; for that, validation and followreaction monitoring assays on the authors note, but the latter is difficult to up experiments using orthogonal company’s QTRAP system. Still, he minimize. methods are necessary. says, “Two-hundred-fifty-plus publicaThe team also identified a third con“The advantage of iTRAQ is [it tions [using iTRAQ] cannot be wrong.” founding factor: contamination of the handles] eight biological samples at Dana-Farber Cancer Institute reiTRAQ m/z 121 channel (m/z 121.12) once, and that’s a very good thing to searcher Jarrod Marto, who uses both with [M+1] immonium ions (m/z have,” he says. “But the downside is iTRAQ and TMT reagents in his lab, 121.08) from phenylalanine-containing [that] the quantitation is perhaps not says he has experienced the lowball peptides. as accurate as you would like.” figures that isobaric reagents can re“I think iTRAQ is a very, very good —Jeffrey M. Perkel port and applauds Wright for applying tool, but I think you need to take the
10.1021/pr900897d
© 2009 American Chemical Society
Journal of Proteome Research • Vol. 8, No. 11, 2009 4885
