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Targeted proteomic approaches for detection of CD-specific epitopes T. AMERICA (1), H. van den Broeck (1), J. Cordewener (1), L. Gilissen (1), I. van der Meer (1) (1) Plant Sciences Group - Wageningen UR, Wageningen, Netherlands.
Proteomics analysis comprises the detailed characterization of protein composition of biological extracts. LC-MS, liquid chromatography on-line coupled to mass spectrometry, is able to deliver an extremely detailed inventory of the protein extracts. So-called non-targeted analysis will generate protein lists per sample, containing identity and quantity information. <i>Non-targeted</i> proteomics analysis is the method of choice for discovering quantitative differences between sample groups, in order to discover relevant changes in protein composition and select for marker peptides that can be used for subsequent targeted detection analysis. The targeted approach focusses on accurate and sensitive quantitative detection of selected peptides for which specific detection procedures are developed. We have developed a targeted detection assay for epitope-specific a-gliadin peptides that are known to be the major toxic epitopes involved in celiac disease (CD). CD-patients that have to maintain their gluten-free diet are dependent on reliable testing and labeling of gluten-free products. Detection of gluten proteins containing CD-epitopes is affected by the extraction protocol and the accuracy of the detection method. So far, the R5-ELISA is the approved method to detect if food products can be labelled gluten-free. The R5-ELISA makes use of monoclonal antibody that detects gluten in general and does not specifically detect CD stimulating epitopes. Recent research has demonstrated that the CD-immunogenicity varies across the variety of gluten proteins among and within different wheat varieties and species. Our targeted LC-MS MRM method can detect quantitatively and simultaneously 9 different a-gliadin peptides, of which 7 contain CD-inducing epitopes. We have tested our method by analyzing several wheat varieties that vary in CD-epitope content. View Presentation |
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