Beyond gluten proteomics: accessibility of sidechains in the gluten network

There have been many attempts at untangling the maze of flexible protein molecules that make up the gluten network in grains, flours, dough, and in cereal-based products through a number of distinct (and ever-changing) interactions. The time-proven use of selective/conditional solubility approaches prior to proteomics analysis often may be inappropriate for structural studies, as these approaches rely almost invariably on some denaturing steps of different severity, that disturb or destroy intra- and interprotein interactions. We report here on using specific reagents, capable to evaluate the exposure of suitable "reporter" residues under more or less stringent network-loosening conditions (presence of detergents or chaotropes, or temperature, for instance). In particular, single- and double-labeling techniques were used to label with distinct fluorophores the protein thiols exposed under various conditions, allowing a preliminary proteomics analysis of the mutual relationships among proteins in grains and in foods and of their possible changes upon processing. We also report on attempts at using gold nanoparticles for similar "conditional accessibility" studies, taking advantage of the remarkable thiol reactivity of the surface of gold nanoparticles and of the resistance of the covalent thiol-gold interaction to the most common network-dissociating agents. Some preliminary "gluten interactomics" data were collected, by using MS-based proteomics approaches and by exploiting the possibility of "pulling" from the gluten network both those proteins that expose gold-reactive sites and those that are interacting with the gold-captured proteins through either disulfide or non-covalent bonds, without establishing a direct interaction with the gold nanoparticles themselves.