PEPTIDE PROFILING IN CHEESE SAMPLES BY LC-TANDEM MASS SPECTROMETRY

Liquid Chromatography coupled with Mass Spectrometry has been extensively used to analyze intact milk proteins or peptides released from milk proteins (1). Nevertheless, the characterization of proteolytic profile in dairy products is still challenging since it is the result of a complex series of events coming from non-specific enzymatic systems, as well as technological food processing. In shot-gun proteomics experiments, the computational analysis of MS/MS data is the real “Achilles heels” (2) and different methods for assigning peptide sequences to MS/MS spectra are critically discussed and compared (3). In this work, the water-soluble oligopeptide fractions of fiordilatte and cream cheese during refrigerated storage was monitored by nanoLC and electrospray ionization ion trap mass spectrometry coupled with a bioinformatics approach based on the scoring distribution and a post database search validation. MS/MS Ions Search was performed by using MASCOT in-house and consecutively setting as enzyme choices, the most active proteases in animal species (trypsin, pepsin, chymotrypsin, trypsin+chymotrypsin) in addition to the option “none” and, finally, semitrypsin. The peptide sequence assignments were cross checked by the evaluation of score, rank, number of experimental product ions and their relative abundances in the MS/MS spectrum. Only peptides present in the list of the protein hits were considered for the sequence identification by processing both the entire chromatographic run and selected time-windows corresponding to the most intense peaks. The proposed method allows the characterization of the most relevant peptides and minimizes the risk of false-positive identifications, reducing analysis time and costs, with the advantage of obtaining important information by a single LC-MS/MS run. A dynamic evolution of hydrophilic peptides was observed belonging mainly to β-casein and -casein in fiordilatte and cream cheese, respectively. Characteristic trends with maximum and minimum values at different times were observed, throughout the refrigerated storage, leading to continuous changes in the peptide composition. [1] S. Sforza, V. Cavatorta, F. Lambertini, G. Galaverna, A. Dossena, R. Marchelli, J. Dairy Sci. 95 (2012) 3514-3526. [2] S.D. Patterson, Nat. Biotechnol. 21 (2003) 221-222. [3] A.I. Nesvizhskii, J. Proteomics 73 (2010) 2092-2123