From proteins to bread: novel tools for the molecular description of protein-­protein interactions (and their evolution) in baked products

Cereal­-based matrices are deeply studied and characterized from a rheological and technological point of view. Although protein-­protein interactions at the base of gluten formation are well known (i.e. disulfide exchange reactions and hydrophobic interactions), the peculiar chemistry of the proteins involved in the formation of the network - gliadins and glutenins - is still a source of discussion. The present work aims at developing new molecular approaches suitable to describe the “thiolome” of the system, in order to provide information on gluten protein structure and protein-­protein interactions during processing. The thiolomic approach relies on labeling accessible free cysteine thiols with a specific iodoacetamide derivative fluorescent probe, in the presence of different destructuring agents (SDS and urea). Labeled proteins are separated in mono-­ and/or bi­-dimensional electrophoresis. Image analysis is used to compare the fluorescent emission map and the total protein pattern to detect changes in the distribution of free thiols among the various proteins. The comparison of the information obtained from the thiolomic approach with those obtained from other approaches, such as front-­face fluorescence and differential solubility, can provide various insights into the evolution of the protein structure of the matrix. Preliminary results on a soft wheat­-based baked product (bread), show that changes in the thiols pattern during kneading - i.e., when disulfide exchange and rearrangement of hydrophobic patches occur - are comparatively modest with respect to those observed after cooking, a step characterized by temperature-­induced structural rearrangements and by water transfer from proteins to starch.