Research And Analysis On Urine Proteomics In Hepatitis B Virus Associated Glomerulonephritis

Objective: To analyse the polypeptide pattern in urine of patients with hepatitis B virus associated glomerulonephritis(HBV-GN) ,and compare with the urine protein of primary membranous nephropathy patients and chronic hepatitis B patients. To screen the protein which has high sensitivity and otherness in the urine of HBV-GN, and to explore the clinical application of mass spectrometry technology for the early noninvasive diagnosis of HBV-GN.Methods: Morning first medistream urine samples from 7 HBV-GN (pathological type is membranous nephropathy) patients and 8 primary membranous nephropathy patients and 7 chronic hepatitis B patients which have not renal damage. To avoid bacterial growth and protein enzymolysis, stored samples at -80℃in 1h. After melting completely, every sample was filtered through 0.22μm D filtration membrane to remove cells debris and other large impurities. Urine samples were fractionated by pre cold acetone precipitation (the volume ratio was 4:1) at - 20℃overnight. Then centrifugated at 4℃4000 x g for 30 minutes, and abandoned supernatant fluid ,and obtained the precipitate which contained urine protein, then dried down in a vacuum centrifuge. After that, the protein concentration of each sample was determined by Bradford (Commassie Blue Fast Staining Solution) assay after dissolved fully with PBS( phsosphate buffer). Put 0.5mg of samples to enzymolysis, then took it to desalt at HPLC. After that, dried and concentrated the liquid mixture which contained peptides. Then went to HPLC-ESI-MS/MS. The protein identification was searched in the database of ipi. human, v3.05 with the help of MASCOT.Results: Through the manual validation, 622 proteins were identified from 7 HBV-GN patients, there were 11 proteins commonly expressed;520 proteins were identified from 8 primary membranous nephropathy patients, and 9 proteins commonly expressed;730 proteins were identified from 7 chronic hepatitis B patients, and 10 proteins commonly expressed. Given intergroup comparison, 5 proteins have the different expression in the urine of HBV-GN patients. They are Alpha-1B-glycoprotein,Ceruloplasmin,Alpha-1-antichymnotrypsin,Ig gamma-2 chain C region,an unmanmed protein (B3KS79);4 proteins have the different expression in the urine of primary membranous nephropathy patients. They are Serotransferrin,Alpha-1-antitrypsin,Ig lambda chain C region,Vitamin D-binding protein. In this experiment , no protein has the different expression in the urine of chronic hepatitis B patients.Conclusion: HPLC-ESI-MS/MS is a quick, easy-handling and high-throughput analytic method, and has its unique advantages in the protein isolation and identification process. Alpha-1B-glycoprotein,Ceruloplasmin,Alpha-1-antichymnotrypsin,Ig gamma-2 chain C region and an unmanmed protein have the different expression in patients with HBV-GN, and they may become biomarkers of hepatitis B virus associated glomerulonephritis.