Pang et al. Clin Proteom (2018) 15:5 https://doi.org/10.1186/s12014-018-9183-3 Clinical Proteomics RESEARCH Open Access Urine proteomics of primary membranous nephropathy using nanoscale liquid chromatography tandem mass spectrometry analysis Lu Pang1, Qianqian Li2, Yan Li2, Yi Liu1, Nan Duan1 and Haixia Li1* Abstract Background: 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. Methods: 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. Results: A total of 509 proteins and 411 proteins were identifed in TMT1 and TMT2, respectively. 249 proteins were both identifed in two technical replicates. GO analysis and KEGG analysis revealed immunization and coagulation were predominantly involved. Among the diferential protein, the overexcretion of alpha-1-antitrypsin (A1AT) and afamin (AFM) were validated by Western blot analysis. Conclusions: 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. Keywords: Primary membranous nephropathy, Urine, Proteomics, Alpha-1-antitrypsin, Afamin Background barrier is injured, leading to massive loss of proteins in Primary membranous nephropathy (PMN) is the most urine (proteinuria) [5]. Histopathologically, this disor- common cause of nephrotic syndrome in the adult popu- der is characterized by discovery of immune complexes lation [1, 2] and it is one of the most common diseases deposits in a specifc part of glomerulus between the glo- afecting the glomerulus [2]. Te kidney fltration barrier merular basement membrane and podocyte (called sub- consists of capillary endothelial cells, glomerular base- epithelium), which contain podocyte antigens or planted ment membrane and highly specialized epithelial cell, antigens and circulating antibodies specifc to those anti- the podocytes [3, 4]. In patients with PMN, this fltration gens, resulting in complement activation [2]. Tis dis- covery corroborates the hypothesis that the most likely pathogenesis of PMN is autoimmune. *Correspondence: [email protected] Tremendous insights into the pathophysiology of 1 Department of Clinical Laboratory, Peking University First Hospital, PMN have been made recently, with studies that have Beijing, China Full list of author information is available at the end of the article identifed M-type phospholipase A2 receptor (PLA2R) © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pang et al. Clin Proteom (2018) 15:5 Page 2 of 15 [6] and thrombospondin type-1 domain-containing providing the preliminary evidence of protein biomarkers 7A (THSD7A) [7] as two major autoantigens in PMN. in urine of PMN patients. Tese fnding supported the theory that podocytes act as sources of antigens for the formation of subepithelial Methods immune complexes deposits [8]. Serum anti-PLA2R anti- Sample collection body identifes approximately 60–80% of cases of PMN Midstream morning urine samples were collected from [9–11]. Despite the undeniable potential of anti-PLA2R patients of biopsy proven PMN with positive anti-PLA2R antibody as a biomarker of disease in patients with PMN, antibody (Group A, n 32) and negative anti-PLA2R this antibody does not explain the etiology of the disease = antibody (Group B, n = 31). Patient characteristics are in a substantial proportion of cases [12]. shown in Additional fle 1: Table S1 and Additional fle 2: Despite progress in the understanding of pathogen- Table S2. Cases of secondary MN were excluded from the esis, the diagnosis of PMN is defnitively dependent on present study, in particular patients with systemic auto- only renal biopsy [13]. Te exact pathogenesis of PMN immunity diseases, viral hepatitis B and C and HIV infec- remains unknown, but podocytes and podocytes-related tion, neoplastic conditions and exposure to toxic agents. proteins appear to have a pivotal role in the develop- All samples were collected at the time of biopsy. Urine ment of PMN. Te pathogenicity of anti-PLA2R has not samples from healthy volunteers (Group C, n = 32) were yet been confrmed [14]. However, human anti-THSD7A also collected (Additional fle 3: Table S3). Samples were has recently been shown to induce PMN with proteinuria kept for less than 4 h at room temperature followed by in mice [15]. Te morphology of healthy podocyte foot low speed centrifugation at 2000×g, 10 min at room processes is necessary for maintaining the character- temperature to remove cellular debris and then stored at istics of the kidney fltration barrier [16]. Interaction of − 80 °C until use. circulating autoantibodies with antigens at the podocyte At frst, we performed the frst TMT experiment (called cell membrane-basement membrane interface generally TMT1 in this study). To increase accuracy repeatability, is regarded as the fundamental pathological mechanism we performed another TMT experiment (called TMT2 in [17]. Since the visceral epithelial cell of Bowman’s capsule this study) by increasing the sample size and replicates to is podocytes, the content of podocytes will be released validate TMT1 (Table 1). Samples from group A, group into urine when podocytes were destroyed by membrane B and group C (9 patients in each group) were tested attack complex. Terefore, urine can be considered as by Western blot (Additional fle 1: Table S1, Additional a potential source to provide important clues of patho- fle 2: Table S2, Additional fle 3: Table S3). physiological mechanisms of PMN. Tis study was approved by the ethics committee of Nanoscale liquid chromatography tandem mass Peking University First Hospital and informed consents spectrometry analysis (LC–MS/MS) may be consid- were obtained from all participants. ered a novel method for identifying candidate bio- markers for patients sufering from PMN [18]. Tandem Protein precipitation mass tag (TMT) is a label-based quantifcation tech- nology that enables accurate and simultaneous com- Te 10 mL sample aliquots from each participant were parison of multiple samples for protein and peptide thawed, and 2.5 mL of trichloroacetic acid (TCA) quantifcation [19]. TMT coupled with LC–MS/MS has the ability to analyze hundreds of proteins with the high- resolution, mass accuracy, and sensitivity [20]. LC–MS/ Table 1 Flow chart of experimental design with three MS has been applied in the proteomic analysis of vari- biological replicates and two technical replicates ous kidney diseases, such as acute kidney injury [21], Replicates Groups Number TMT tags lupus nephritis [22], diabetic nephropathy [23] and IgA of samples nephropathy [24]. However, proteomic analysis of PMN TMT1 Group A 5 126 has been rarely studied, especially in urine. Group B 4 130 Our aim was to test urine proteomics as a non-invasive Group C 5 131 method for identifcation of new protein biomarkers of TMT2 PMN in urine, and link them to pathogenesis of the dis- TMT2a Group A 9 126 ease through known signaling and metabolic pathways. Group B 9 127C In this study, we performed proteomic analysis using Group C 9 127 N TMT technology coupled with LC–MS/MS, compar- TMT2b Group A 9 128 N ing PMN urine samples with normal control groups. Group B 9 129C Te proteomic results were validated with Western blot, Group C 9 130C Pang et al. Clin Proteom (2018) 15:5 Page 3 of 15 precipitation solution (30% v/v) were added to the sam- subjected to a matrix assisted laser desorption ionization ple aliquots to a fnal TCA concentration of 6% v/v. procedure after Ziptip desalting. Strong vortexing for 1 min and overnight incubation at Strong cationic exchange chromatography − 20 °C were performed. Te mixtures were centrifuged at 14,000×g for 15 min at 4 °C and the supernatants Te mixed peptides were dissolved in bufer A (2% ace- were discarded. In order to eliminate traces of acid that tonitrile (ACN) and 20 mmol/L ammonium formate, can negatively afect the digestion efciency, the pellets pH 10.0). Ten, the samples were loaded onto a reverse- were resuspended in 2.5 mL of chilled acetone (− 20 °C) phase column (Luna C18, 4.6 × 150 mm; Phenomenex, and clarifed by centrifugation at 14,000×g at 25 °C for Torrance, CA, USA) and eluted using a step linear elution 10 min. Te wash step was repeated once more. Te program: 0–10% bufer B (500 mmol/L KCl, 10 mmol/L supernatants were discarded and the pellets were dried KH2PO4 in 25% ACN, pH 2.7) for 10 min, 10–20% bufer naturally. Te dry pellets were resuspended in 300 μL B for 25 min, 20–45% bufer B for 5 min and 50–100% solubilization bufer (8 mol/L urea and 0.1 mol/L ammo- bufer B for 5 min at a fow rate of 0.7 mL/min.