Preliminary analysis of urinary proteome of children minimal change nephrotic syndrome by twoª²dimensional gel electrophoresis and PDQuest software based image analysis

    HUANG Yanª²Jun£¬HUANG Songª²Ming£¬ZHANG Aiª²Hua£¬ FEI Li£¬XU Songª²Zhou£¬CHEN Rongª²Hua

    Center of Paediatric Nephrology£¬Nanjing Medical University£¬Nanjing 210029£¬China

    ¡¾Abstract¡¿ AIM: To analyze the urinary proteome of children minimal change nephrotic syndrome by the twoª²dimensional gel electrophoresis (2DGE) and PDQUEST software based image analysis. METHODS: The urinary protein of children nephrotic syndrome was isolated by cold acetone precipitation, treated to remove high abundance proteins by Aurum Serum Protein Kit and desalted by ReadyPrepTM2ª²D Cleanup Kit. The rehydrated immobilized pH gradient (IPG) strips containing protein samples were isoelectric focusing (IEF). After IEF, the equilibrated strips were transferred to the vertical SDSª²polyacrylamide gel. The silver stained gels were scanned by UMAX PowerLook 1120 densitometer to acquire the digitalized images. Using new generation twoª²dimensional image analysis software, PDQuest 7.3, the 2DGE of proteins extracted from urine was processed. The analysis procedure, including image acquirement, automated spot detection and quantification and analysis result report, was carried out. The method and the volume of loading sample, the choice of IPG gels, the concentration of SDS gels and the protocol for staining procedure were improved. RESULTS: Using the proper method stated above, satisfactory 2DGE maps of urinary protein were obtained and the preliminary analysis results were reported. The reasonable sample load and Voltª²hours of urinary protein were about 100 ¦Ìg and 60 000 Voltª²hours for the 17 cm pH3ª²10 IPG strips. A total of 400 protein spots were visualized with PDQuest 7.3 software. CONCLUSION: Urinary proteomics can be applied in the study of urine protein of children minimal change nephrotic syndrome. We have used proteomic method to construct protein maps of urinary protein of children minimal change nephritic syndrome. At the level of proteome, we may use urinary proteomic techniques to find functional proteins and specific proteins (protein biomarker) of renal disease in the future and the technique will be further used in renal research to characterize network mechanisms of physiological processes and the disease. ......