Comprehensive profiling of post translational modifications (PTMs) on processed bovine whey proteins

This work has shown that data evaluation settings have a significant impact on the results of untargeted post translational modification(PTM) profiling. Therefore, further research should carefully adjust the software parameters to enable the most comprehensive PTM profiling. It was also shown that the identification results are subject to high variation for multiple measurements of the same sample. It was concluded that samples should be analyzed in triplicate and it is recommended that an identification should only be accepted if a peptide is identified in at least two out of three measurements. This should substantially reduce the risk of false positive identifications. Based on the presented method, it should be feasible to extend PTM profiling to other milk proteins. The comparison of an untargeted PTM profiling method with a targeted method showed that a targeted approach has a sensitivity advantage, making it a valid approach for a pre-selected list of modified peptides. However, the untargeted method was able to identify far more PTMs on far more modified peptides. Another advantage of the untargeted method is the possibility to re-evaluate the data with different settings. For example, the acquired data could be analyzed with an extended selection of PTMs. The applied untargeted PTM profiling method was able to reflect the heat exposure different whey protein samples well for up to four days. However, PTM monitoring in raw milk whey showed a different pattern compared to the other sample types. The formation of PTMs was notably lower in the whey samples. Further studies on the effect of whey constituents such as vitamins on PTM formation are needed to understand this observed difference between whey and the other samples.