Application of Two-Dimensional Gel Electrophoresis To Interrogate Alterations in the Proteome of Genetically Modified Crops. 1. Assessing Analytical Validation

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• 作者：Martin C. Ruebelt ; Nancy K. Leimgruber ; Markus Lipp ; Tracey L. Reynolds ; Margaret A. Nemeth ; James D. Astwood ; Karl-Heinz Engel ; and Klaus-Dieter Jany
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2006
• 出版时间：March 22, 2006
• 年：2006
• 卷：54
• 期：6
• 页码：2154 - 2161
• 全文大小：300K
• 年卷期：v.54,no.6(March 22, 2006)
• ISSN：1520-5118

文摘

Current tools used to assess the safety of food and feed derived from modern biotechnologyemphasize the investigation of possible unintended effects caused directly by the expression oftransgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomicevaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Becauseanalytical technologies are rapidly developing, proteome analysis based on two-dimensional gelelectrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DEmethod was established for the qualitative and quantitative analysis of the seed proteome ofArabidopsis thaliana with the following validation parameters examined: (1) source and scope ofvariation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolvesproteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and issensitive enough to detect protein levels in the low nanogram range. The separation of the proteinswas demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI andMM directions, respectively. The mean coefficient of variation of 254 matched spot qualities wasfound to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R ² >0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majorityof selected proteins. Therefore, this method could be used to interrogate proteome alterations suchas a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point,and/or quantity of a protein.Keywords: Proteomics; two-dimensional gel electrophoresis; validation; unintended effect; seedproteome; Arabidopsis