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An In-Depth Analysis of Proteomics Expression Profiling in Rat Glomeruli Utilizing LC-MS

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An In-Depth Analysis of Proteomics Expression Profiling in Rat Glomeruli Utilizing LC-MS Articles Preclinical Medicine July 2010 Vol.55 No.20: 2142–2151 doi: 10.1007/s11434-010-3291-4 SPECIAL TOPICS: An in-depth analysis of proteomics expression profiling in rat glomeruli utilizing LC-MS HONG Quan1*, XUE Peng2*, LÜ Yang1, CHEN XiangMei1, QI Ka1 & WU Di1† 1 Kidney Department & Institute of Nephrology, Division of Clinical Internal Medicine, Chinese PLA General Hospital, Beijing 100853, China 2 Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Received December 23, 2009; accepted April 8, 2010 Glomeruli are an essential functional element of renal filtration. The majority of renal diseases caused by glomerular sclerosis or fibrosis may result in renal dysfunction. A fomulate protein profile, a comprehensive analysis of glomeruli of normal rats was conducted in this study via protein spectrum. Functional annotation and classification of these proteins were performed and it was found that 26 had the same glomerule (endothelial cells, podocytes and mesangial cells) markers with proteins. glomeruli, protein spectrum, multidimensional protein identification technology, glomerule marker, homologene Citation: Hong Q, Xue P, Lü Y, et al. An in-depth analysis of proteomics expression profiling in rat glomeruli utilizing LC-MS. Chinese Sci Bull, 2010, 55: 2142−2151, doi: 10.1007/s11434-010-3291-4 Glomeruli are a critical element of the kidneys, and func- disease (CDK) have proposed to analyze the proteins in the tions’ urine filtration. Glomerular dysfunction due to scle- urine, which may represent the plasma protein instead of rosis or fibrosis is a common cause of the end stages of re- native proteins of kidneys in terms of the abnormal tubule nal diseases. The molecular pathogenesis mechanism of infiltration, secretion and reabsorption. Few studies had renal diseases associated with glomerular sclerosis or fibro- been carried out by proteomic analysis based on the anat- sis, remain uncertain. A variety of proteins may be involved omy of kidneys. Zhao et al. [1] described the proteome pro- in the initiation and development of glomerular sclerosis or file of the renal cortex of mice, which was characterized by fibrosis. Therefore, it is necessary to examine the proteome 1967 kinds of proteins, most of which were consistent with profile of glomeruli at the physiological level, which may the protein identified in urine. By using one-dimension significantly contribute to understanding the machinery of separation (SDS-PAGE), two-dimension separation (IEF- the development of glomeruli sclerosis or fibrosis. SDS) and liquid chromatography-mass spectrometry (LC- The proteomic profile of kidneys has been investigated MS), Miyamoto et al. [2] found 6686 kinds of proteins in related to chronic disease of kidneys (CDK), because it is the glomeruli of normal renal tissue from a urethral carci- able to provide information about pattern switch of proteins noma patient. However, proteomic studies with kidney dis- expression in kidney as the process of CDK, and might po- ease in rats, as the primary model, have rarely been reported. tentially identify key proteins or drug targets which trigger Multi-dimension protein identification technology (MDP- or represent events. A relatively complete proteome data- IT) was developed to analyze the proteome by Yates et al. base of biomarkers in CDK has been developed by the Hu- [3]. The protein complex is cleaved into polypeptide frag- man Kidney and Urine Proteome Project which login ments by certain proteases and the polypeptide fragments in 2005. Most investigations focused on chronic kidney are analyzed and identified by cationic exchange and re- verse-phase two-dimension LC/MS. The previous methods, *These authors contributed equally to this work including SDS-PAGE and IEF-SDS, separate the protein in †Corresponding author (email: [email protected]) the gel, subsequently isolate and assay the polypeptide © Science China Press and Springer-Verlag Berlin Heidelberg 2010 csb.scichina.com www.springerlink.com HONG Quan, et al. Chinese Sci Bull July (2010) Vol.55 No.20 2143 fragments. MDPIT, compared with the latter two ap- The tissue blocks were sectioned and dewaxed. Periodic acid- proaches, may identify the low-abundance or hydrophobic Schiff stain was applied to observe the tissue pathology, with membrane proteins, and proteins with a broader isoelectric images captured by a microscope with a camera. point or a low molecular weight. Regarding the advantages of MDPIT, in this study, it was 1.4 Purification and separation of the glomeruli utilized to detect and identity the protein expression in the normal glomeruli from rats. The results of this study might The glomeruli of normal rats were isolated as described by significantly enhance the understanding of the protein pro- Krakower et al. [4]. The renal cortex was sterilely obtained file related to normal kidney function, and facilitate screen- and cut into thin strips which were grounded, and filtrated ing and targeting proteins, which might alter the expression through an 80-mesh wire sieve. The glomeruli and smudge pattern in the process of CDK, enabling it to clarify the cells below the mesh were collected, with further purifica- pathogenesis of CDK as well. tion of the glomeruli made utilizing a 150-mesh wire sieve. 1 Materials and methods 1.5 Identification of glomerular purity After the collected glomeruli were resuspended in the PBS, 1.1 Subjects one droplet was placed on a glass slide. The purity was ob- The male Sprague Dwaley (SD) rats were purchased from served with a 200× light microscope and images were cap- Vital River Lab Animal Technology Co., Ltd., aged from 8 tured. An appropriate amount of glomeruli were taken and to 10 month, weight from 180 to 200 g. These rats were the cell lysate was achieved utilizing a RIPA buffer (con- bred in a Specific Pathogen Free (SPF) environment with taining 50 mmol/L of pH 7.5 Tris-HCl, 150 mmol/L of free diet and drinking. The urinary sample of 24 h was har- NaCl, 0.5% deoxycholic acid, 1% Nonidet P-40, 0.1% SDS, vested up to 3 d, and a 2-mL urine sample of each animal 1 mmol/L PMSF, a variety of protease inhibitors: 1 μg/mL). was collected for the quantitative measurement of protein After schizolysis, it was placed at room temperature for 15 and systolic blood pressure was monitored and recorded. min, centrifuged at 12000 r/min for 20 min at 4°C. The su- The animals were intraperitoneally injected with 2% pento- pernatant was obtained to determine the concentration barbital sodium (40 mg/kg) for anesthesia, a blood sample within a BCA kit. The protein samples were denatured by was obtained from the abdominal aorta, nephridial tissue SDS, 80 μg protein was run in SDS-PAGE, and transferred was isolated and stored at –80°C. to the PVDF membrane. The membrane was blocked by 5% skim milk in TBST at 4°C overnight, incubated with 1.2 Main reagents E-Cadherin polyclonal antibody with 1:100 dilution (Goat anti-rats, SantaCruz, USA) at room temperature for 4 h, BCA protein concentration detection kit (Bio-Rad, USA), rinsed 3 times by 1 × TBST, incubated with anti-goat HRP iodoacetamide, DTT, ammonium bicarbonate, carbamide, conjugated secondary antibody at room temperature for 1 h. protease inhibitor (Sigma, USA), sequencing grade trypsin Color was developed by an ECL chromogenic system. The (Promega, USA), ultra-pure water produced by the Milli-Q Alpharmimage 2002 system was used for scan and a gray- system (Millipore, USA), acetonitrile and formic acid (JT scale analysis was performed. Baker Phillipsburg, USA), C18 filler (particle size 3.5 μm, bore diameter 120-Å, the Great Eur-Asia Sci & Tech De- 1.6 Preparation of MS protein samples velopment Co., Ltd, China), SCX filler (particle size 5 μm, bore diameter 300 Å, Phenomenex, USA). Purified glomeruli were resuspended by MilliQ ultra-pure water, rinsed 3 times to remove the PBS solution. The sam- ple (800 g) was centrifuged for 5 min to remove the excess 1.3 Monitoring of the basic conditions of rats water. Glomeruli (10 mg) was dissolved in a 1-mL buffer (i) Detection of renal functions. The Bradford method was with 7 mol/L carbamide and 2 mol/L thiocarbamid, was used to detect the protein concentration in urine, which was sonicated, centrifuged, discarding the pellets and retaining multiplied by the urinary production in 24 h to obtain the the supernatant. Protein concentration was determined by 24-h urine production. BCA assay. To break down the disulfide bond, 100 μg of (ii) Detection of systolic blood pressure. An MRB2 III glomerular protein was treated by 10 mmol/L DTT at 56°C A computer rat blood pressure and heart rate meter (pro- for 1 h, cooled down to room temperature, 50 mmol/L IAM duced by the Shanghai Institute of Hypertension) was used was rapidly added to close hydrosulphonyls and the mixture to measure the systolic blood pressure in rat tail arteries. was left undisturbed at room temperature in a dark room for (iii) PAS stain of the nephridial tissues of rats. Nep- 45 min. DTT (40 mmol/L) was added to the sample and the hridial tissue was fixed with 10% formalin overnight, subse- sample was kept at room temperature for 15 min to quench quently dehydrated and lucidificated, embedded in paraffin. the excess IAM. Subsequently, the sample was diluted with 2144 HONG Quan, et al. Chinese Sci Bull July (2010) Vol.55 No.20 25 mmol/L ammonium bicarbonate to 5 times the original transfer tube was 200°C. The mass-spectrometric data was volume. The sample was trypsinized at a 1:1000 ratio at collected in the Data Dependent Acquisition (DDA) model, 37°C for 12 h followed by further trypsinization with the i.e.
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