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UK Team Explores Proteomics-Based Equivalence Testing for Genetically Engineered Tomatoes

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A team led by researchers at the University of London has completed a study using mass spec-based proteomics to assess "substantial equivalence" in genetically engineered crops.

The work, detailed in a paper published in the current edition of the Journal of Experimental Botany, suggests a role for proteomics in the regulation of GE foods, said Peter Bramley, professor of biochemistry at Royal Holloway University of London and an author on the paper.

GE crops like Monsanto's Roundup Ready soya have been available commercially since the mid-1990s. However, such plants remain controversial with consumers, particularly in the EU where the European Food Safety Authority is responsible for regulating their use.

One of the concepts used by the EFSA to regulate GE crops is the idea of "substantial equivalence," meaning that standard regulations can be applied to GE crops provided they are shown to be substantially equivalent in their composition to conventional crops.

Protein analysis is already being used in Europe for such testing "to a limited extent," Bramley told ProteoMonitor this week via email. But "DNA-based techniques are favored in the [food safety] legislation, especially for detection and labeling purposes," he said.

The authors suggest, however, that their findings indicate that mass spec-based shotgun proteomics is "a valid analytical tool for assessing substantial equivalence."

Current testing protocols measure proteins coded by the modified gene or genes, but analysis of the wider proteome is not typically done, Bramley said. As he and his colleagues note, however, previous studies of GE tomatoes have found "significant alterations to the metabolome and transcriptome" of these lines, suggesting the need for a "holistic approach, using all the omic technologies, to judge substantial equivalence between parent and transgenic crops."

To demonstrate the suitability of mass spec for such work, the researchers compared the proteomes of azygous tomatoes, wild type Ailsa Craig tomatoes, and Ailsa Craig tomatoes with the tomato phytoen synthase-1 gene – Psy-1 – in both the sense and antisense orientations.

Psy-1 codes for the protein phytoene synthase, which is a key player in carotenoid biosynthesis, with Psy-1 sense lines exhibiting elevated carotenoid levels and the antisense lines exhibiting a lack of carotenoids.

Using iTRAQ labeling along with strong cation exchange fractionation and nano-LC prior to tandem mass spec analysis on both an Applied Biosystems QSTAR Pulsar I QTOF and an Agilent QTOF 6520, the researchers characterized the proteomes in four biological replicates of each of the tomato cultivars.

They found no significant differences between the technical replicates but significant differences between the various lines in both the total numbers of proteins identified and in their level of expression.

Across two injections, the researchers identified roughly 350 proteins in the wild type Ailsa Craig strain, 300 in the azygous plants, 300 in the Psy-1 antisense tomatoes, and roughly 200 in the Psy-1 sense tomatoes.

The authors speculated that the fewer number of proteins identified in the Psy-1 sense plant could be "a reflection of their smaller size and pericarp volume, due to the phytohormone imbalance in this line, as a consequence of the channeling of isoprenoids from gibberellins to carotenoids."

Bramley reiterated that he believed this result was a reflection of the Psy-1 sense tomato's reduced proteome. However, it could reflect limitations of the mass spec analysis, as well, he said, noting that "it may be that certain proteins are below the threshold for detection by [multidimensional protein identification technology]."

Bramley noted that, as in the case of many organisms, technical issues currently prevent researchers from achieving complete coverage of the tomato proteome.

"No proteomics approach can yet identify, never mind quantify, a [full] plant proteome," he said. "The coverage will improve as the technology becomes more mature, as databases expand, and more genomes are sequenced."

"So, at present, predicted [protein] changes can be quantified, plus a subset of other proteins that come from MudPIT [analysis]," he added.

Beyond the technological limitations, proteomics will need to overcome skepticism from regulatory agencies like the EFSA, Bramley said. "As with all new technologies [regulatory agencies] will need to be convinced that such an approach is robust, reproducible, gives added value, and can be undertaken by public analysts."

Cost is a concern, as well, he added, noting that mass spec proteomics is relatively expensive with respect to reagents and equipment.

With regard to that last issue, however, Bramley said that he and his colleagues have just completed a study on using label-free quantitative proteomics to analyze GE tomato fruit.

"This avoids the use of iTRAQ and so is much less expensive for routine use," he said.

In addition to GE "substantial equivalence" testing, mass spec-based proteomics is making its way into food testing in a variety of areas with the hope of improving on existing tests and anticipating future testing needs (PM 1/13/2012).

Major vendors including Bruker and AB Sciex have begun expanding beyond small molecule-based testing into protein-based applications including detection of biopolymer contaminants, food mislabeling, and microbial detection.

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