Identification of Marker Peptides for the Whey Protein Quantification in Edam-Type Cheese

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides ('bottom-up' proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG). Based on the findings, alpha-LA and beta-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R-2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% +/- 0.02% to 5.31% +/- 0.25% for beta-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

Automated summary

The aim of this study was to develop a LC-MS/MS method for quantifying individual whey proteins and validate it using a model cheese. Alpha-lactalbumin and beta-lactoglobulin were found to be resistant to proteolytic degradation during ripening, with no effect on the marker peptides. Most marker peptides showed good linearity, repeatability, and recovery rate. However, absolute quantification revealed differences in whey protein content depending on the marker peptide, suggesting further research is needed for valid quantification in different cheese types.