Qualitative and quantitative analysis of mixed-milk cheeses by targeted proteomics

This work presents a novel analytical method to determine the proportions of cow, sheep and goat milk in mixed-milk cheeses, common in Spain and Europe. First, an in-depth shotgun proteomic analysis to define the protein composition of milk and cheese from each species was performed. Thereafter, a targeted parallel reaction monitoring method was developed using a panel of 3 species-specific tryptic peptides and one peptide common to all species. This method was optimized using reference cheeses of known composition and it was validated according to internationally recognized guidelines. The method demonstrated linearity in the range of = 0% to 100% with detection limits less than 1% for all 3 species and showed good repeatability (CV = 8%), reproducibility (CV = 10%) and accuracy (99.6%), enabling both qualitative and quantitative analysis. Finally, the method was applied to commercial cheeses with diverse compositions and ripening times. The measurements were unaffected by either ripening or production process, demonstrating the robustness of the analysis even with potential interferents. These results demonstrate the great potential of the method for quality control purposes and its applicability to real-world commercial samples.