Bottom-up proteomics to investigate the X-ray irradiation effects on soft cheese

Food irradiation is an alternative technological process employed to preserve the hygienic quality of food and to extend its shelf life. In this work, three different types of soft cheese, mozzarella, robiola and primosale, were treated with X-rays at 1.0, 2.0 and 3.0 kGy to evaluate their effect on proteins and peptides using a bottom-up proteomic approach. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF-MS) and liquid chromatography coupled with MS through electrospray interface (LC-ESI-MS) were employed to analyse the peptide mixtures coming from the proteolytic digestion of soft cheese using both trypsin and chymotrypsin enzymes. We discovered 15 peptides most affected by X-rays that were sequenced by tandem MS and identified; ten of these discriminant peptides, generated from the cleavage of β-casein and α-casein (B. taurus), were localized on the proteins' secondary structure, highlighting a heightened susceptibility of random coils to X-ray irradiation. No additional post-translational modifications were found to be induced or emphasized by irradiation. For all studied cheese samples, the major spectral differences were encountered at 1.0 kGy, suggesting that such a dose was the most effective in promoting peptide bond cleavage. Some non-enzymatic and/or half-enzymatic peptides belonging to α-S1-, α-S2- and β-casein were also produced when varying X-ray doses.