Investigation of immunogenic gluten peptides: Identification using enzymatic tagging and HPLC-MSn analysis and quantification using HPLC-MS/MS.

摘要

The goal of my research was to provide some insight into a widely appreciated but poorly understood relationship between cereal grain proteins and human health. My research objectives were: (1) to identify and characterize inflammatory, physiologically relevant, wheat gluten peptides; and (2) to develop a unique analytical methodology to screen commercially available food and consumer products for the quantitative detection of these peptides.;Gluten proteins comprise a very large protein family found in cereal grain seeds. This large protein family consists of hundreds of proteins ranging in size from about 30 kDa into the millions of KDa. Today's nomenclature refers to "gluten" as the water-insoluble seed storage proteins found in the Triticeae tribe of the grass (Gramineae) family that includes wheat, rye and barley. Some gluten proteins associated with grains in the Triticeae tribe (specifically: wheat, rye and barley), have been implicated in various autoimmune diseases, food allergies, intolerances and are important factors in several inflammatory diseases.;Many analytical techniques have been used to study gluten proteins and peptides. Unambiguous identification and structural characterization of such peptides is a necessary step toward an eventual understanding of their chemical biology. Liquid chromatography-mass spectrometry (LC-MS) is an extremely powerful tool for such analyses.;Results of my research are presented here, in the following chapters of this thesis report. Data presented supports the development of a novel and effective analytical methodology, using enzymatic/chemical labeling chemistry and HPLC/MSn; to identify and characterize seven physiologically relevant wheat gluten peptides. A sensitive and specific assay was then developed for the quantitative detection of these peptides via direct in-vitro proteolytic digestion and HPLC-MS/MS. This versatile methodology allows both processed and native foods, as well as consumer products, to be analyzed for the presence of wheat gluten. Continued efforts in this area will pave the way for eventual commercial application, as a service to both the celiac community and the food industry, by providing an accurate and economic means to generate much needed data for researchers developing treatments for patients with gluten sensitivities and manufacturers producing and labeling products that are safe.