Unravelling elusive mysteries of bovine milk proteins by mass spectrometry: From colostrum to mature milk, during processing and throughout storage

Milk is the first source of nutrition of the neonate and has constituted a major food group in the human diet for over 9 millennia, also beyond infancy. The discovery of milk proteins dates back to the early 1800s, marking over 2 centuries of milk protein research. More recently, genome sequencing and the advancement of mass spectrometry techniques have created the opportunity for booming research on milk proteins. The hereby generated knowledge is part of the foundation for understanding the physical-chemical and biological functionality of milk proteins. The work described in this PhD thesis is based on modern mass spectrometric approaches applied for studying the dynamic changes in bovine milk proteins, with emphasis on carbohydrate modifications of proteins including glycation and glycosylation. Glycation is a chemical modification that alters the digestibility and decreases the bioavailability of essential amino acids from milk proteins. Here, we found that milk protein glycation was triggered during the thermal processing steps during production and advanced during subsequent storage of skim milk powder. We characterized the diversifying milk proteoform profiles, with identification of glycation hot-spots, and of a preferential primary sequence motif of glycation. Glycosylation is an enzymatic modification of the proteins, which heavily impacts a multitude of biological processes and functions. Our study of N-glycosylation during the transition from colostrum to mature milk in individual cows revealed changes in (N-glyco)protein abundances and dynamic changes in protein-specific N-glycosylation during lactation. Colostrum and mature milk proteins exhibited distinct N-glycosylation characteristics, indicating differences in their origin and functionality.