cow MFGM LC-MS/MS - Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane

Pasteurized whole cow milk (Grade A) was purchased from the supermarket. Whole cow milk was centrifuged at 3 000 g at 4°C for 30 min and the fat layer was recovered. The recovered fat was washed with PBS for 20 min at 37°C, and the resuspened mixture was centrifuged again to obtain the milk fat. The wash procedure was repeated three times. Finally, milk fat was washed with MilliQ water to recover the fat globules. Milk fat globules were solubilized with a SDS solution (100 mM Tris-HCl, pH 7.4, 100 mM DTT and 4% SDS) and then incubated in 95°C water bath for 10 min. The mixtures were centrifuged at 14 000 g for 15 min, and the protein samples were collected. S-Trap, FASP, in-gel, and traditional Chl/MeOH, as well as modified chloroform/methanol (Chl/MeOH) or acetone precipitation followed by in-solution digestion without clean-up of tryptic peptides methods were applied to characterization cow MFGM proteome.