Integrating the information from proteomic approaches: A “thiolomics” approach to assess the role of thiols in protein-based networks Stefania Iametti a, ⁎, Mauro Marengo a , Matteo Miriani a , M. Ambrogina Pagani b , Alessandra Marti b , Francesco Bonomi a a Section of Chemistry and Biomolecular Sciences, Italy b Section of Science and Technology of Food Systems, DeFENS, University of Milan, Via G. Celoria, 2, I-20133 Milan, Italy abstract article info Article history: Received 11 October 2012 Accepted 28 December 2012 Keywords: Food proteins Thiols Fluorescent labeling 2D-electrophoresis Denaturing agents Thiol–disulfide exchange reactions, are major contributors to the formation of a covalently-linked protein network in many foods, where disulfides represent the most “natural” type of interprotein covalent bond. Thiol–disulfide exchange reactions occur also as a function of the accessibility of the involved thiols, which in turn depends on structural features of the involved proteins. Thiols in soluble and insoluble food proteins were covalently labeled by 5-iodoacetamide-fluorescein in the absence or in the presence of 4 M urea, a pro- cedure that allowed to evaluate thiols accessibility before and after protein unfolding and dissociation of non-covalently linked protein complexes. Proteins labeled under either condition, along with unlabeled pro- teins, were then solubilized by treatment with disulfide reductants (and urea, when not added before) and separated either by SDS-PAGE or by two-dimensional electrophoresis. The 5-iodoacetamidofluorescein label- ing procedures were also applied to soft wheat flours, and to semolina from durum wheat. Results highlight the different accessibilities of thiols in specific protein components in these materials, suggesting a possible role of minor protein components as for promoting rearrangement in the thiol pattern in wheat proteins upon processing and pointing out the relevance of structural issues in addition to compositional ones. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Cysteine thiols and cysteine disulfides represent the most “natural” way for generating covalently-linked protein networks in the most di- verse foods. Thiol–disulfide exchange events are involved in a range of process-dependent molecular transformations in systems that range from whey proteins to egg proteins, and include water insoluble pro- teins such as those present in cereals. The network-forming capacity of proteins involved in thiol–disulfide exchange reactions in individual food systems is related to a multiplicity of factors, that include their rel- ative abundance, the amount (and location) of reactive thiols and disulfides, and their availability to exchange events. Some of these pa- rameters may be sensitive to process-induced structural modifications of the involved proteins, that may lead to exposure of reactive thiols or to their burial inside the structure of individual proteins and of pro- tein aggregates (Iametti, Cairoli, De Gregori, & Bonomi, 1995; Iametti, De Gregori, Vecchio, & Bonomi, 1996). Structural modifications leading to exposure/burial of potentially reactive thiols have been addressed as a function of physical and chemical denaturation in a rather ample col- lection of soluble food proteins (Iametti et al., 1996, 1999). However, systems comprising water insoluble proteins (such as those in many cereals) are much more difficult to address, although their investigation is of paramount practical and economical significance. The unique properties of proteins in wheat (and in related cereals) are instrumental to the production of extremely diverse common foods. The ability to form a viscoelastic network called gluten among gli- adins and glutelins (Belton, 1999; Gobaa, Bancel, Branlard, Kleijer, & Stamp, 2008; Shewry, Tatham, Forde, Kreis, & Niflin, 1986) is quintessen- tial to the consumer appreciation of the final product, be that due to re- tention of gas bubbles in bread and baked products or to entrapment of swollen starch in pasta (Singh & MacRitchie, 2001). From a molecular standpoint, the interactions leading to the formation of the visco-elastic network of gluten involve rearrangement of hydrophobic contacts among proteins (or within individual proteins) and rearrangement of intra- and intermolecular disulfides and thiols in a disulfide exchange process that requires protein flexibility (provided by the addition of water) and the action of shear forces that act as “mechanical denatur- ants” during mixing (Morel, Redl, & Guilbert, 2002). Gliadins are charac- terized by having mostly intramolecular disulfides, whereas glutelins form large aggregates linked by intermolecular disulfides (Shewry, Halford, Belton, & Tatham, 2002). Given the relevance of disulfides and thiols in these processes, chemical and biochemical oxidants and reductants have often been used as “ameliorants” of dough rheology (Lagrain, Brijs, & Delcour, 2006). Oxidants (such as bromate/iodate or hydrogen peroxide, that may also be produced “in situ by appropriate enzymes (Hanft & Food Research International 54 (2013) 980–987 ⁎ Corresponding author at: DeFENS, Via G. Celoria, 2, I-20133 Milan, Italy. Tel.: +39 02 5031 6819; fax: +39 02 5031 6801. E-mail address: stefania.iametti@unimi.it (S. Iametti). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.12.054 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres