Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation

Abstract

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganismsthat is stored in specialized glands located in the tegument. For some animals, such glands have accumulated inspecific regions of the body and formed prominent structures known as macroglands. The Bufonidae familydisplays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to beextremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though.Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a pro-teomic study on the parotoid macrogland secretion of the Asian bufonidDuttaphrynus melanostictus. By em-ploying the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions -bound and unbound–that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteinsrelated to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases,lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological ac-tivity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present inthe bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played bysuch molecules.Significance:The current approach allowed a detailed proteomic analysis of the several proteins synthesized intheD. melanostictusparotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within acomplex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological roleplayed by such proteins at the skin