EXPLORATION OF THE “HIDDEN” PROTEOME OF DONKEY’S MILK

It is well known that Cleopatra and other privileged ladies of ancient times were taking their bath in donkey's milk (DM) in order to keep their skin fresh and shiny. In addition, Hippocrates as well as Pliny the Elder believed that donkey's milk could act therapeutically in numerous cases, such as liver problems, infectious diseases, fevers, asthma, etc. Nowadays, several studies have clearly demonstrated that DM may be very useful in the prevention of atherosclerosis, being a strong vasodilator and an effective antimicrobial agent,1 and represents a valid alternative to cow's milk proteins allergy.2 Consequently, DM has recently aroused scientific and clinical interest, above all among pediatric allergologists. Notwithstanding this renewed growing interest, until today genome mapping of the Equus genus is largely uncompleted and the characterization at molecular level of DM only concerns the major protein components,3,4,5 whereas nothing is known about the minor or trace protein components. Here, we report the application of the ProteoMiner technology aimed at the investigation of the “hidden” proteome of DM. This technology is based on the capture of all proteins present in biological samples, and the normalization of their concentration via a combinatorial library of hexameric peptide ligands, used as baits for adsorbing all macromolecular species present in solution. 6,7 Protein identification was performed by coupling the ProteoMiner technology, SDS-PAGE, nLC-nESI MSMS and database searching via MASCOT engine. This approach allowed discovering and identifying a total of 106 protein components, most of which were not described in previous proteomic studies, and therefore represent the most comprehensive list of donkey’s whey proteins at present. Seventy percent of identified proteins were by homology with Equus caballus, whereas only 11 proteins (10%) were from Equus asinus. The low number of identified proteins from Equus asinus, can be explained by the low number of databases entries from this species, whose genome is still unsequenced. On the other hand, the large presence of known homologous proteins from the closest related specie (Equus caballus), allowed the cross-species identification for most of the proteins investigated.