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      Environmental Effects on Allergen Levels in Commercially Grown Non-Genetically Modified Soybeans: Assessing Variation Across North America

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          Abstract

          Soybean ( Glycine max) is a hugely valuable soft commodity that generates tens of billions of dollars annually. This value is due in part to the balanced composition of the seed which is roughly 1:2:2 oil, starch, and protein by weight. In turn, the seeds have many uses with various derivatives appearing broadly in processed food products. As is true with many edible seeds, soybeans contain proteins that are anti-nutritional factors and allergens. Soybean, along with milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, and wheat, elicit a majority of food allergy reactions in the United States. Soybean seed composition can be affected by breeding, and environmental conditions (e.g., temperature, moisture, insect/pathogen load, and/or soil nutrient levels). The objective of this study was to evaluate the influence of genotype and environment on allergen and anti-nutritional proteins in soybean. To address genetic and environmental effects, four varieties of non-GM soybeans were grown in six geographically distinct regions of North America (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania). Absolute quantification of proteins by mass spectrometry can be achieved with a technique called multiple reaction monitoring (MRM), during which signals from an endogenous protein are compared to those from a synthetic heavy-labeled internal standard. Using MRM, eight allergens were absolutely quantified for each variety in each environment. Statistical analyses show that for most allergens, the effects of environment far outweigh the differences between varieties brought about by breeding.

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          Most cited references36

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          The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications.

          Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.
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            Does trypsin cut before proline?

            Trypsin is the most commonly used enzyme in mass spectrometry for protein digestion with high substrate specificity. Many peptide identification algorithms incorporate these specificity rules as filtering criteria. A generally accepted "Keil rule" is that trypsin cleaves next to arginine or lysine, but not before proline. Since this rule was derived two decades ago based on a small number of experimentally confirmed cleavages, we decided to re-examine it using 14.5 million tandem spectra (2 orders of magnitude increase in the number of observed tryptic cleavages). Our analysis revealed a surprisingly large number of cleavages before proline. We examine several hypotheses to explain these cleavages and argue that trypsin specificity rules used in peptide identification algorithms should be modified to "legitimatize" cleavages before proline. Our approach can be applied to analyze any protease, and we further argue that specificity rules for other enzymes should also be re-evaluated based on statistical evidence derived from large MS/MS data sets.
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              Two-dimensional gel electrophoresis in proteomics: Past, present and future.

              Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned. Copyright © 2010 Elsevier B.V. All rights reserved.
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                Author and article information

                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Research Foundation
                1664-462X
                27 August 2012
                2012
                : 3
                : 196
                Affiliations
                [1] 1simpleInterdisciplinary Plant Group, Department of Biochemistry, Christopher S. Bond Life Science Center, University of Missouri Columbia, MO, USA
                [2] 2simplePioneer Hi-Bred International Ankeny, IA, USA
                [3] 3simpleDuPont Agricultural Biotechnology, Pioneer Hi-Bred Wilmington, DE, USA
                Author notes

                Edited by: Paul Andrew Haynes, Macquarie University, Australia

                Reviewed by: Bronwyn Jane Barkla, Universidad Nacional Autónoma de México, Mexico; Benjamin Schwessinger, University of California Davis, USA; Roque Bru-Martinez, Universidad de Alicante, Spain

                *Correspondence: Gregory S. Ladics, DuPont Agricultural Biotechnology, Pioneer Hi-Bred, Wilmington, DE 19880, USA. e-mail: gregory.s.ladics@ 123456usa.dupont.com

                This article was submitted to Frontiers in Plant Proteomics, a specialty of Frontiers in Plant Science.

                Article
                10.3389/fpls.2012.00196
                3427918
                22969785
                4d6f89d3-eadb-4d25-840b-3c6f74d3ec91
                Copyright © 2012 Stevenson, Woods, Hong, Kong, Thelen and Ladics.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

                History
                : 28 March 2012
                : 08 August 2012
                Page count
                Figures: 6, Tables: 3, Equations: 2, References: 43, Pages: 13, Words: 9324
                Categories
                Plant Science
                Original Research

                Plant science & Botany
                gmo,glycinin,lc-ms/ms,mrm,allergens,soybean
                Plant science & Botany
                gmo, glycinin, lc-ms/ms, mrm, allergens, soybean

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