Isolated milk fat globules were subjected to enzyme hydrolysis by a specific protease (trypsin) and a nonspecific protease (pronase E) to study the asymmetric arrangement of milk fat globule membrane (MFGM) proteins. The remaining proteins on the globules after proteolysis were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. By this proteomic approach, the results confirmed different susceptibility of the MFGM proteins to proteolysis by enzymes. Butyrophilin and adipophilin were completely digested by trypsin and by pronase E, whereas lactadherin and xanthine dehydrogenase/oxidase were almost resistant to hydrolysis by trypsin and partially attacked by pronase E. Based on our results and recent bibliographic data, an up-dated model of the organization of some MFGM proteins is proposed and discussed. (c) 2011 Elsevier Ltd. All rights reserved.