Peptidomics on two in vitro dynamic digested dairy matrices

Dietary proteins are known to generate through their digestion a large number of different peptides and some of them have shown to play different physiological roles. Therefore, understanding the way food proteins will be disintegrated during digestion process is a major issue. However, identification of peptides in food matrices is a challenging task, and active peptides can be minor constituents in highly complex mixture of peptides. In this context, mass spectrometry has emerged as an important tool for protein identification since it allows the peptide sequencing in mixtures that contain up to hundreds of different peptides without the need of purification. This work aims the identification of major peptides generated after in vitro digestion was performed on two different milk samples (raw and heat treated milk) under dynamic conditions. The samples were analyzed by HPLC-MS/MS, (ion trap and MALDI-TOF/TOF) prior and after digestion, at different digestion times. As expected, the size of peptides lowers down as the digestion process goes on. At early digestion times, the protein coverage is lower than that obtained at later digestion times due to the low solubility of long fragments. At short digestion times, identified β-CN peptides belonged to the carboxy-terminal domain, however, αS1-CN peptides belonged to both, amino- and carboxy-terminal. Comparison of the two matrices reveals that β-CN was more susceptible to be hydrolysed by pepsine in raw than in heat treated milk. As the time of digestion increases, the protein coverage improves, except for the phosphorylated regions for which low ionization was achieved, and therefore they are difficult to identify unless an enrichment protocol is followed before analysis. Among the sequences identified at the end of the gastric digestion (405 min), some of them had been previously reported to exert an antihypertensive effect (for instance, peptide AYFYPEL)1, peptide YFYPEL is able to stimulate mucin secretion and expression in human intestinal cells2, and RYLGYL has been described as opioid.