Application of LC‐HRMS identified marker peptides in an LC‐MS/MS method for detection and quantification of heat‐resistant proteolytic activity in raw milk

For the dairy industry, it would be useful to be able to determine the heat-resistant proteolytic activity of the raw milk upon arrival at the plant, preferably before processing (within 24 h of arrival). This would allow producers to choose the most optimal processing of the raw milk and hence its fate, together with a more accurate prediction of shelf-life. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of Pseudomonas-derived heat-resistant proteolytic activity in raw milk based on three new marker peptides, identified using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), was developed. This method involved a sample preparation where existing peptides are removed from the milk sample, after which the heat-resistant protease-containing fraction is mixed with a casein substrate. After incubation, the increase of the peptide markers is determined using an LC-MS/MS method. The limit of detection (LOD) and limit of quantification (LOQ) of this method were determined using synthetically produced peptides (with an average LOD between 0.1 and 5 ng/mL and an average LOQ between 0.1 and 10 ng/mL).