Urine proteomics of primary membranous nephropathy using nanoscale liquid chromatography tandem mass spectrometry analysis

Lu Pang, Qianqian Li, Yan Li, Yi Liu, Nan Duan, Haixia Li

Primary membranous nephropathy (PMN) is an important cause of nephrotic syndrome in adults. Urine proteome may provide important clues of pathophysiological mechanisms in PMN. In the current study, we analyzed and compared the proteome of urine from patients with PMN and normal controls. We performed two technical replicates (TMT1 and TMT2) to analyze and compare the urine proteome from patients with PMN and normal controls by tandem mass tag (TMT) technology coupled with nanoscale liquid chromatography tandem mass spectrometry analysis (LC-MS/MS). Gene ontology (GO) enrichment analysis was performed to analyse general characterization of the proteins. The proteins were also matched against the database of Kyoto Encyclopedia of Genes and Genomes (KEGG). For validation, Western blot was used to analyze the selected proteins. A total of 509 proteins and 411 proteins were identified in TMT1 and TMT2, respectively. 249 proteins were both identified in two technical replicates. GO analysis and KEGG analysis revealed immunization and coagulation were predominantly involved. Among the differential protein, the overexcretion of alpha-1-antitrypsin (A1AT) and afamin (AFM) were validated by Western blot analysis. Our data showed the important role of immunologic mechanism in the development of PMN, and the value of urinary A1AT and AFM in biomarker discovery of patients with PMN. The discovery of the overexcretion of A1AT and AFM in the urine can help to further elucidate pathogenetic mechanisms involved in PMN.