Primary Study On Urine Proteome In Idiopathic Membranous Nephropathy

Objective:To analyse the polypeptide pattern in urine of patitents with idiopathic membranous nephropathy, and compare with the healthy controls.Aim to find the protein which has high sensitivity and specificity, and to discuss whether this technology is suitable for clinical diagnosis.Methods:Morning first medistream urine samples from three healthy people and nine patients were collected.In order to avoid bacterial growth and protein enzymolysis,stored samples at-80℃during 1h.After melting, every sample was filtered through 0.22μmD filtration membrane to remove impurities.Urine samples were frationated by acetone precipitation(the volume ratio was 4:1)at-20℃overnight.Then centrifugated at 4℃4000xg for 20 min and obtained the precipitate which contained urine protein, dried down in a vacuum centrifuge. The protein concentration of each sample was determined by Bradford assay. The protein was digested with trypsin overnight at 37℃.Then went to HPLC-ESI-MS/MS. The protein identifi-cation was searched in the database of ipi.human,v3.05with the help of MASCOT.Result:101 proteins were identificated from healthy people.There were 37 proteins commonly expressed.179 proteins were identificated from the urine of patitents with idiopathic membranous nephropathy.There were 20 proteins commonly expressed.Compare with healthy controls,8 protein specifically expressed in patitents with idiopathic membranous nephropathy. They are Ceruloplasmin,Ig lambda chain C region,Alpha-1-acid glycoprotein,Vitamin D-binding protein,Leucine-rich alpha-2-glycoprotein,ATP-sensitive inward rectifier potassium channel 11,Calcium-binding protein 39,IPI00745619(Conserved hypothetical protein). Conclusion:We used HPLC-ESI-MS/MS to analyse the polypeptide pattern in urine of patitents with idiopathic membranous nephropathy.174 protein were identificated.Ceruloplasmin,Alpha-1-acid glycoprotein,Vitamin D-binding protein,Leucine-rich alpha-2-glycoprotein,Calciun-binding protein 39 may become biomarkers of idiopathic membra-nous nephropathy.