Observational Study On Urine Glycosylated Proteomics In Immunoglobulin A Nephropathy With Uyghur Nationality

Objective: To research urine glycosylated proteomics spectrum characteristics in initial diagnosis patients of primary immunoglobulin A nephropathy with Uyghur nationality, and compare with the healthy people. In order to observe the liquid chromatography combined with mass spectrometry in Uyghur Ig A nephropathy and to explore the clinical diagnosis value in the disease.Methods: In 2013 December to 2014 August to take morning first medistream urine specimens with newly diagnosed by renal biopsy in patients with 20 primary Uygur Ig A nephropathy and 10 healthy people at same times in the People’s Hospital of Xinjiang Uygur Autonomous Region. And immediately joined the hydrochloric acid into the samples for embaming. In order to avoid bacterial contamination and protein enzymolysis in within 2 hours at 4000g(4 celsius) for 10 min centrifugation to remove cell debris and related components, collected the supernatant. And placed in the refrigerator to save at-80 celsius. Then remove the 30 urine samples, and dissolved completely to room temperature. In accordance with the specimen volume: acetone volume at 3: 1ratio rapidly added-20 celsius precooled acetone to precipitate protein. Then centrifugated the samples again at 4 celsius 14000 g for 30 min and obtained the precipitate which contained urine protein, dried down in a vacuum centeifuge and to store in the-80 celsius for reserving. The urine protein precipitate after vacuum drying with PBS solution fully dissolved. The protein concentration of each urinary sample was determined by Bradford method. Application of hydrazide chemistry enrich glycoproteins and PNGase F enzyme, dried and concentrated in vacuum. Then went to two dimensional liquid chromatography-tandem mass spectrometry proteomic techniques on a sample protein group carried on the system analysis, and obtained the differential expression protein. We used Mascot software for data retrieval to obtain polypeptide sequence results, and searched urine protein sequence in the Uniprot database to achieve query specific expression of protein names, molecular mass, isoelectric point, cellular localization and other related information.Results:(1) 121 glycoproteins from 10 Uyghur healthy human urine samples were identified after validation, all healthy human urine samples commonly expressed 23 proteins.(2) 218 glycoproteins were identified from the urine of 20 patients with Uyghur immunoglobulin A nephropathy urine samples. 17 proteins commonly expression of all samples in immunoglobulin A nephropathy. Compared with urine proteins from healthy human, there were 6proteins differentially expressed in immunoglobulin A nephropathy. They are Alpha-l-antitrypsin,Ceruloplasmin, Serotransferrin, Vitamin D binding protein, Alpha-l microglobulin and Alpha-amylase 1. In these proteins, which α1- microglobulin downregulated in immunoglobulin A nephropathy, other proteins are upregulated.Conclusion: We used two dimensional liquid chromatography-tandem mass spectrometry technology to research on the urine protein mass spectrometry characteristics of immunoglobulin A nephropathy patients in Uyghur people. And obtained polypeptide sequence spectrum of the disease successfully. 218 glycoproteins were identificated. There were 6 proteins differentially identificated in immunoglobulin A nephropathy with Uyghur nationality. Alpha-l-antitrypsin,Ceruloplasmin, Serotransferrin, Vitamin D binding protein, Alpha-l microglobulin and Alpha-amylase 1 may become biomarkers of Immunoglobulin A nephropathy in Uyghur people.