It is difficult for membrane proteins to get into 2-D gel because of their hydrophobic properties and the contaminations of carbohydrates in protein samples also affect the results of 2-D gel. The modified 2-D electrophoresis technique described in this paper overcomes these two difficulties, and have successfully separated proteins from sucrose gradient ultracentrifugation and got membrane proteins into 2-D gel. Proteomic analysis was then performed on proteins from prevacuolar compartment. Around 200 protein spots were separated on the 2-D gel, 23 protein spots were cut out form the gel and subjected to MS/MS analysis. After database searching, 13 proteins did not hit matching proteins, with the 10 proteins that hit matching proteins, 4 of them have known protein function, 6 of which turned out to be unknown or unnamed proteins.