Protein Characterization by MALDI In-Source Decay Mass Spectrometry in Support of Safety Assessments of Genetically Modified Crops.

The advancement of mass spectrometry provides advantages for transgenic protein characterization in support of safety assessments of genetically modified crops. Here, we describe how matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) in combination with intact mass and bottom-up analyses can be applied to achieve high confidence in the sequences of transgenic proteins expressed in plants and establish the biochemical equivalence of microbially produced protein surrogates. ISD confirmed 40-60 near terminal residues regardless of the protein size, including the improvement of the coverage of cysteine-rich proteins by the reduction/alkylation of disulfide bonds.

Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098.

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure.

Background: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios.

Aim: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability.

When the Whole is Not Greater than the Sum of the Parts: A Critical Review of Laboratory Bioassay Effects Testing for Insecticidal Protein Interactions.

Many studies have been conducted to investigate synergism among insecticidal proteins; however, a consensus on minimal data requirements and interpretation is lacking. While some have concluded that all additive predictive-type models should be abandoned, we advocate that additivity models can remain useful as assessment tools and that an appropriately designed interaction study will never systematically underestimate the existence of synergism, irrespective of which additivity model (or none at all) may be used. To generate the most meaningful synergy assessment datasets in support of safety assessments, we highlight two beneficial steps to follow: (i) select a testing model which is the most consistent with current knowledge regarding the action of the insecticidal proteins and (ii) avoid using bioassay methods which may result in excess response heterogeneity.

A General Approach to Test for Interaction Among Mixtures of Insecticidal Proteins Which Target Different Orders of Insect Pests.

A shift toward transgenic crops which produce combinations of insecticidal proteins has increased the interest (Syngenta Seeds, Inc., Minnetonka, MN) in studying the potential for interactions amongst those proteins. We present a general testing method which accommodates proteins with nonoverlapping spectrums of activity.

A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops.

Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet.

Characteristics and safety assessment of intractable proteins in genetically modified crops.

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins.

Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop.

Utilization of the genetic resources of wild species to create a nontransgenic high flavonoid tomato.

Flavonoids represent a large and important group of plant natural products that are ubiquitous in the plant kingdom. Epidemiological studies have shown the health benefits of a diet high in flavonoids. However, the dietary intake of flavonoids in most western populations is limited, creating a need to find alternative food sources for these polyphenolic secondary metabolites.

Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites.

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro.

Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa.

Two UDP-glucose-dependent flavonoid glucosyltransferases (EC 2.4.1.