A simple and rapid procedure was cl developed for the extraction of the milk fat globule membrane from milk removes the large majority of the skim milk proteins for proteome analysis. In order to improve the extraction and the solubilization of the hydrophobic membrane proteins for subsequent two-dimensional gel electrophoresis, four detergents (3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate, amidosulfobetaine-14, sodium lauroyl sarcosinate and sodium deoxycholate) were tested in the sample preparation, associated with a sonication step. Zwitterionic detergents were shown to be efficient in recovering integral and peripheral proteins from membrane material. Spots were identified by matrix-assisted laser desorption/ionization tandem-time-of-flight (MALDI-TOF/TOF). The advantages of MALDI-TOF/TOF (speed, easiness of analysis, good sensitivity and high mass accuracy) were demonstrated on the milk fat globule membrane proteome investigation. Identified proteins are implicated in a wide range of functions including fat secretion and transport, protein trafficking and regulation. (C) 2008 Elsevier Ltd. All rights reserved. ; Peer reviewed