Combining the reactivity of gold nanoparticles and proteomics approaches to address protein-protein interactions in food proteins

The thiol-binding capacity of Au nanoparticles (AuNPs, 20 nm) was used to test accessibility of protein thiols in wheat flour, where proteins interact through both hydrophobic interactions and disulfide bonds. At difference with soluble thiol reagents, the AuNPs size limits their penetration in protein networks. Also, the stability of the thiol ligation to AuNPs simplifies removal of unbound proteins and recovery of AuNPs (and of covalently bound proteins) through centrifugation, making it possible to identify AuNPs-bound proteins through MS/MS and limited proteolysis. Low concentrations of detergents allowed controlled breakdown of hydrophobic interactions and allowed to distinguish binding of AuNPs to insoluble network-forming proteins rather than to soluble ones. Both glutenins (having free cysteines and intramolecular disulfides) and gliadins (no free thiols and only intramolecular disulfides) were found to be covalently bound to AuNPs, proving that the two protein classes form intermolecular disulfides already in the flour. These results pave the way to further studies on the time course and the molecular determinants of protein-protein interactions during deposition in the seed or on the role of some proteins or protein classes in gluten formation. Use of AuNPs of different size should also make it possible to assess the geometry and the structure of the protein network in foods, either wheat-based or not.