Large variation in bovine milk composition of Dutch Holstein cows has been observed. The factors influencing the milk synthesis and secretion process in the mammary gland and the variations in this process lead to variation in milk composition. The understanding of milk synthesis was improved during the last decades, however, much is still unknown, especially with regard to lipid synthesis and secretion. In this research, a proteomics technique (FASP-Dimethyl labeling-NanoLC-Orbitrap-MS/MS) was used to identify and quantify low abundant proteins in milk. By using this proteomics technique combined with a metabolomics technique (1H-NMR), the variation in milk synthesis and secretion processes were shown as affected by several factors influencing milk composition: DGAT1 polymorphism, fat globule size and energy balance of the cow. Stomatin, cholesterol and cholesterol synthesis enzymes were shown to be important for milk synthesis and secretion in lactating cows in all the different studies. Cytoplasmic vesicle and cytoskeleton proteins were shown to be involved in milk fat globule secretion. These results led to new insights in milk synthesis and secretion.