Primary Study On Urine Protein In IgA Nephropathy With HPLC-MS/MS

Objective :To study urine protein character of mass spectrometry in patitents with Immunoglobulin A nephropathy, which combine with kidney puncturation ,and compare the mass spectrometry pictures of different types. It is aim to find the good specificity and sensitivity protein, and to debate whether the technology is suitable for clinical diagnosis.Methods: 8 morning first urine was obtained from 7 patients ,and avoided to bacteria and protein enzymolysis, spun at 3000×g for 5min at 4℃to remove cells, and took the supernatant filtrationed from 0.45μm D filter membrane to remove big matters. Freezed acetone was Pulled into every sample, with the volume ratio was4:1, stored at-20℃for a night. Then centrifugated at 4℃7000×g for 30 min,and obtained the precipitate which contained urine protein, then dryed and concentrated at -106℃.After that,it was dissolved completely with PBS and measured by Bradford testing method, and get the protein concentration. Putting 0.8g of samples to enzymolysis. Before taking it to desalt at HPLC, adjusted it to 0.8μg/μl.The fluid which cotained peptides was obtained, then dryed and concentrated at -106℃,then went to LC-MS/MS(LCQ).Setting m/z for the second MS at the range of 400~2000.The whole time was 210mins,The time of cleaning the machine was 30 mins for each sample.The protein identification was performed using SEQUEST algorithm incorporated in BioWorks software,and searched in the database of ipi.human,v3.05.Results: Satisfactory MS patterns of urine proteins were obtained, and discovered 194 types of protein .There were 15 proteins specifically expressed in these different pathological types of IgA nephropathy. The proteins were identificated at the database of International Protein Index (IPI)human, and found 13 ones.There were 28 proteins found in the urine of focal and segmental glomerulosclerosis (LeeⅢ),and get 36 proteins in focal proliferative glomerulosclerosis (LeeⅡ). Although the quantity of urine protein was less than before treated by glucocorticoid (LeeⅠ), the number of protein was not decreased in midrange meseng proliferative. The same protein were 36 types before and after treatment.Conclusion: The technology of LC-MS/MS was used to separated and analyzed urine proteomics in the patient of IgA nephropathy. Satis- factory MS patterns of urine proteins were obtained, and discovered 194 types of protein,most of these proteins were glucoprotein.The Ig excreting fragment,HP protein and Uteroglobin may be regarded as biomarks of IgA nephropathy .