The present study aimed the evaluation of saliva sample pre-treatment, in particular the sample clearance usually performed by centrifugation, to the contribution of salivary proteome and peptidome. Using in-gel and off-gel approaches, a large content of salivary proteins was detected in the pellet fraction that is usually discarded. In addition, chaotropic/detergent treatment in combination with sonication, before the centrifugation step, resulted in salivary complex disruption and consequently in the extraction of high amounts of proteins. Based on this data, we suggest the use of urea/detergent with sonication as a standard saliva sample pre-treatment procedure. We also described a procedure to extract salivary peptides which can be performed even after saliva sample treatment with chaotropic/detergents. In overall, we reported for the first time the contribution of the pellet fraction to the whole saliva proteome. iTRAQ analysis highlighted a higher number of different peptides as well as distinct quantities of each protein class when after sample treatment with urea and sonication, acetone precipitation followed by solubilization with acetonitrile/HCl was performed.

In recent years, several studies have demonstrated that polyunsaturated fatty acids have strong immunomodulatory properties, altering several functions of macrophages. In the present work, we sought to provide a multi-omic approach combining the analysis of the lipidome, the proteome, and the metabolome of RAW 264.7 macrophages supplemented with phospholipids containing omega-3 (PC 18:0/22:6; ω3-PC) or omega-6 (PC 18:0/20:4; ω6-PC) fatty acids, alone and in the presence of lipopolysaccharide (LPS). Supplementation of macrophages with ω3 and ω6 phospholipids plus LPS produced a significant reprogramming of the proteome of macrophages and amplified the immune response; it also promoted the expression of anti-inflammatory proteins (e.g., pleckstrin). Supplementation with the ω3-PC and ω6-PC induced significant changes in the lipidome, with a marked increase in lipid species linked to the inflammatory response, attributed to several pro-inflammatory signalling pathways (e.g., LPCs) but also to the pro-resolving effect of inflammation (e.g., PIs). Finally, the metabolomic analysis demonstrated that supplementation with ω3-PC and ω6-PC induced the expression of several metabolites with a pronounced inflammatory and anti-inflammatory effect (e.g., succinate). Overall, our data show that supplementation of macrophages with ω3-PC and ω6-PC effectively modulates the lipidome, proteome, and metabolome of these immune cells, affecting several metabolic pathways involved in the immune response that are triggered by inflammation.

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.