In-gel protein N- and C-termini identification and its application for transgenic protein characterization.

A new method for the determination of N- and C-termini of a protein isolated in a polyacrylamide gel is introduced. In-gel partial protein hydrolysis by hydrochloric acid is used to generate N- and C-terminal peptides for identification. This new method is complementary to existing techniques. The application of the in-gel protein termini identification technique to the characterization of the transgenic protein diacylglycerol acyltransferase (UrDGAT2A) purified from soybean seeds is also reported here. Both N- and C-termini of UrDGAT2A were successfully identified and the N-terminus was found to be blocked by acetylation. The analysis results of UrDGAT2A and two commercial proteins (bovine serum albumin (BSA) and alcohol dehydrogenase) are used to demonstrate the effectiveness of the method in identifying actual N- and C-termini, terminal truncation and blocking.

Metabolite analyses of grain from maize hybrids grown in the United States under drought and watered conditions during the 2002 field season.

Understanding natural variation in the composition of conventional crop germplasms is critical in establishing a baseline for comparison of biotechnology-derived crops. This is particularly relevant to such traits as tolerance to drought stress. Thus, there is both a need to understand the contribution of stress conditions to natural variation in plant nutritional components and to determine whether levels of small molecule metabolites such as osmoprotectants and stress metabolites are also affected. As a first step in developing such information for maize, seven conventional hybrids were grown under different moisture regimens and the impact of moisture on composition was assessed. The regimens included well-watered conditions, water restriction during the vegetative phase, and water restriction during grain fill. Compositional analyses of the harvested grain included assessments of the levels of proximates (moisture, protein, oil, starch) and small molecule metabolites such as fatty acids, free amino acids, organic acids, sugars, total glycerol, glycine betaine, and abscisic acid. Ranges for these analytes were determined across all moisture regimens, and the effect of the different water regimens on these analytes was also evaluated. The number and type of grain analytes that showed statistically significant differences in levels between different water regimens differed quite markedly by maize hybrid. However, the magnitude of mean differences between well-watered and water-restricted samples was typically small, and statistically significant differences for any given analyte were typically observed in only one to three of the seven maize hybrids. Only two analytes, free glutamine and free proline, showed a significant drought-induced difference in at least four maize hybrids.

Impact of genetics and environment on nutritional and metabolite components of maize grain.

The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnology-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnology-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly associated with a newly introduced trait. Therefore, steps have been initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against two different testers. These were grown at three different, but geographically similar, locations in the United States. In addition to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, organic acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant organic acid was citric acid, with only minor amounts of other organic acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and organic acids appeared to be markedly higher than that observed for the OECD analytes.

Application of (1)h NMR profiling to assess seed metabolomic diversity. A case study on a soybean era population.

(1)H NMR spectroscopy offers advantages in metabolite quantitation and platform robustness when applied in food metabolomics studies. This paper provides a (1)H NMR-based assessment of seed metabolomic diversity in conventional and glyphosate-resistant genetically modified (GM) soybean from a genetic lineage representing ∼35 years of breeding and differing yield potential. (1)H NMR profiling of harvested seed allowed quantitation of 27 metabolites, including free amino acids, sugars, and organic acids, as well as choline, O-acetylcholine, dimethylamine, trigonelline, and p-cresol. Data were analyzed by canonical discriminant analysis (CDA) and principal variance component analysis (PVCA). Results demonstrated that (1)H NMR spectroscopy was effective in highlighting variation in metabolite levels in the genetically diverse sample set presented. The results also confirmed that metabolite variability is influenced by selective breeding and environment, but not genetic modification. Therefore, metabolite variability is an integral part of crop improvement that has occurred for decades and is associated with a history of safe use.

Identification of plant sphingolipid desaturases using chromatography and mass spectrometry.

This manuscript describes an efficient analytical assay combining high-performance liquid chromatography with UV detection (HPLC-UV), liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS), and gas chromatography with mass spectrometry (GC-MS) for the characterization and C=C bond localization on the long chain base of sphingolipids in yeast extracts in order to identify the plant sphingolipid desaturases activity. Samples of wild type control and transgenic yeast expressing putative sphingolipid desaturases were hydrolyzed into long chain bases. Mono-unsaturated long chain base, dehydrophytosphingosine (t18:1), in transgenic yeast as a result of the function of plant sphingolipid desaturase was detected with cis, trans-isomers resolution by reverse phase HPLC-UV as DNP (2,4-dinitrophenyl) derivatives along with saturated phytosphingosine (t18:0). The molecular structure of phytosphingosine was confirmed by negative-ion LC-MS-MS, which also served as a rapid tool for screening the plant spingolipid desaturase activity with 2-min run time under multiple-reaction monitoring (MRM) mode. The C=C bond location of dehydrophytosphingosine was further identified by GC-MS after being converted into picolinyl derivatives. This assay combines multiple chromatographic and mass spectrometric techniques with gentle chemical procedures to provide capacities for rapid determination of the plant sphingolipid desaturase activity as well as identification of their active sites in the backbone of the sphingolipid species in yeast.