Study of the oligopeptide fraction in Grana Padano and Parmigiano-Reggiano cheeses by liquid chromatography electrospray ionization mass spectrometry

The oligopeptide fractions of Grana Padano and Parmigiano Reggiano cheese samples, at various stages of ageing (6, 12, 18 and 24 months), were analysed by means of high-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS). The main oligopeptides (< 5000 Da) present in the samples were extracted by using an originally developed method which allowed good enrichment of the oligopeptide fractions and identified according to their molecular weights. The casein sequences compatible with the found molecular weights were determined and the exact oligopeptide sequences were identified by using the fragmentation peaks originated by in-source collisionally-induced dissociation (CID). The ESI-MS method reported here allowed the sequence identification of oligopeptides very quickly with a single liquid chromatography-mass spectrometry run. Oligopeptides were also semi-quantified by comparison with a suitable internal standard (the dipeptide phenylalanyl-phenylalanine, Phe-Phe). This methodology demonstrated that, as far as the main oligopeptides are concerned, Grana Padano and Parmigiano Reggiano presented very similar composition. Anyway, the evolution of the fractions during ageing time was characteristically distinct among the two cheeses: in Grana Padano cheeses most of the oligopeptides reached a maximum at 12 months of ageing and then decreased, whereas in Parmigiano Reggiano cheeses the oligopeptide amounts were usually lower and had a less regular trend. The reason for this different behaviour may be ascribed to the different production techniques.