Modern proteomic methodologies for the characterization of lactosylation protein targets in milk

Heat treatment of milk induces the Maillard reaction between lactose and proteins; in this context, beta-lactoglobulin and alpha-lactalbumin adducts have been used as markers to monitor milk quality. Since some milk proteins have been reported as essential for the delivery of microelements and, being resistant against proteolysis in the gastrointestinal tact, also contributing to the acquired immune response against pathogens and the stimulation of cellular proliferation, it is crucial to systematically determine the milk subproteome affected by the Maillard reaction for a careful evaluation of aliment functional properties. This is more important when milk is the unique nutritional source, as in infant diet. To this purpose, a combination of proteomic procedures based on analyte capture by combinatorial peptide ligand libraries, selective trapping of lactosylated peptides by m-aminophenylboronic acid-agarose chromatography and collision-induced dissociation and electron transfer dissociation MS was used for systematic identification of the lactosylated proteins in milk samples subjected to different thermal treatments. An exhaustive modification of proteins was observed in milk powdered preparations for infant nutrition. Globally, this approach allowed the identification of 271 non-redundant modification sites in 33 milk proteins, which also included low-abundance components involved in nutrient delivery, defence response against virus/microorganisms and cellular proliferative events. A comparison of the modified peptide identification percentages resulting from electron transfer dissociation or collision-induced dissociation fragmentation spectra confirmed the first activation mode as most advantageous for the analysis of lactosylated proteins. Nutritional, biological and toxicological consequences of these findings are discussed on the basis of the recent literature on this subject, emphasizing their impact on newborn diet.