Maize roots release a battery of proteins into the rhizosphere during growth and development. In the present study, mucilage secreted by primary roots of 3-4 d old maize seedlings was collected under axenic conditions, and the constitutively secreted proteins were analyzed by different methods, including two-dimensional electrophoresis--MADLI-TOF, LC-MS, and Shotgun techniques. The results showed that 200 protein spots could be observed on two-dimensional electrophoresis gel stained by silver nitrate. However, due to the low protein content for each protein spot, there was not reliable result after identification by MADLI-TOF. On the other hand, 152 and 2848 proteins were identified by LC/MS and Shotgun technique, respectively. All 152 proteins identified by LC/MS method could be found in the proteins identified by Shotgun method, and thus the results obtained by LC/MS and Shotgun method could be authenticated mutually. The results demonstrated that the Shotgun method is more sensitive than the other methods for protein identification, especially for the proteins with lower contents and complex interfering substances, such as the plant root released proteins.