scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Western Blot ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses can not. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results. The identified proteins use GO analysis for subcellular localization, KEGG analysis for functional classification and signal pathways, STRING analysis for functional protein association networks.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism.Conclusions: Compared with H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2 virus.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Western Blot ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background In the past 20–30 years, both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses could cause severe damage and a high death rate, but H9N2 viruses could not. In this study, we use H9N2 virus infection as a control to investigate the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections in A549 cells. Methods According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results, and then perform GO and KEGG analysis. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Conclusions Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism and the cytoskeleton. The differential expression of eight mitochondrial proteins in H5N1-infected cells resulted in decreased T cell activation, decreased antigen presentation and stress response, reduced ATP synthesis, and decreased induction of apoptosis, resulting in the higher pathogenicity of H5N1 virus than H9N2 virus.These finding may provide a basis for analyzing the pathogenesis of influenza viruses with different virulence levels, identifying anti-influenza host targets and developing new influenza vaccines.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. Methods According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. Conclusions Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9 

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses cannot. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections. Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected,, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, STRING analysis for functional protein association network construction. Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots further mass spectrometry identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis and metabolism. Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2021 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection (MOI) virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism.Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

A Proteomic Approach to Discovery of Candidate Proteins Involved in the Transformation of Follicular Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 984-984
Author(s):  
Guang Fan ◽  
Yanping Zhong ◽  
Cristina Smith ◽  
James Huang ◽  
Rita Braziel
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Gel Electrophoresis ◽  
Western Blot Analysis ◽  
Protein Identification ◽  
Differentially Expressed ◽  
Signal Transduction Pathways ◽  
Differentially Expressed Proteins ◽  
2D Gel ◽  
2D Gel Analysis

Abstract Background: Follicular lymphoma (FL) undergoes transformation to a high grade diffuse large B-cell lymphoma (tr-DLBCL) in about 50% of patients. During transformation, a more virulent subclone of tumor cells emerges, leading to a rapidly progressive clinical course and resistance to therapy. The identification of proteins involved in transformation is critical for understanding the mechanism of transformation and developing molecularly targeted therapy. In this study, we compared protein expression between grade 1- FL (G1-FL) and tr-DLBCL using 2D-gel electrophoresis and Western blot analysis. Design: Frozen tissue and frozen cells were obtained from the Department of Pathology, Oregon Health and Science University tumor bank. The protein expression profiles of 3 G1-FL and 3 tr-DLBCL were compared using 2D-gel electrophoresis. Protein identification was done using a MALDI mass spectrometer. Frozen cells of an additional 11 non-paired GI-FL and 11 non-paired tr-DLBCL, and 2 pairs of G1-FL and tr-DLBCL specimens were used for Western blot confirmation of the initial 2D-gel findings. Results: 2D-gel analysis and MALDI protein identification revealed 14 differentially expressed proteins between G1-FL and tr-DLBCL (figure 1), all of which are known to play important roles in cellular energy/metabolic pathways, signal transduction pathways, and protein and nuclear synthesis. The two most differentially expressed proteins on 2D-gel analysis were superoxide dismutase (MnSOD2) and growth factor receptor bound protein 2 (Grb2). Western blot analysis of MnSOD2 and Grb2 confirmed their relative over- or under-expression in frozen cells from multiple additional clinical lymphoma samples, including 2 paired- and 22 non-paired G1-FL and tr-DLBCL. Both 2D-gel analysis and Western Blot showed a significantly higher level of expression of MnSOD2 and a lower expression of Grb2 expression in tr-DLBCL (figure 2). Summary: Using proteomic profiling, confirmed by Western blot analysis of clinical G1-FL and tr-DLBCL samples, we have confirmed 2 proteins (MnSOD2 and Grb2) that are expressed at significantly different levels in G1-FL and DLBCL. MnSOD2 is capable of protecting cells from reactive oxygen species and regulating signal transduction pathways to influence cell growth and apoptosis. Inhibition of MnSOD2 has been shown in studies of several cancer cell lines to render cancer cells more susceptible to apoptosis. Grb2 is a member of a critical signaling pathway leading to Ras activation in hematopoietic cells. Both proteins may play a critical role in FL transformation. These proteins have the potential to be therapeutic drug targets, diagnostic and/or prognostic markers, or biomarkers for monitoring therapeutic response. Summary of Differentially Expressed Spots Summary of Differentially Expressed Spots Figure Figure

Proteomic Signature Predicting Achievement of Very Good Partial Response In Patients with Multiple Myeloma Based On Complementary Label-Free and iTRAQ Quantitative Proteome Analysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1902-1902
Author(s):  
Dominik Dytfeld ◽  
Malathi Kandarpa ◽  
John R Strahler ◽  
Dattatreya Mellacheruvu ◽  
Suchitra Subramani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Western Blot ◽  
Differential Expression ◽  
Proteomic Analysis ◽  
Quantitative Proteomics ◽  
Proteomic Profile ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Rt Pcr

Abstract Abstract 1902 Introduction: Multiple myeloma (MM) remains mostly incurable. Novel therapies have improved response rates, which are now reaching 100%. More importantly, number of recent studies showed that the depth of response, e.g. achievement of at least 90% reduction of the disease (≥VGPR) is associated with longer disease control. Therefore, improving VGPR rates and establishing predictors of VGPR to a given regimen may be an important clinical goal. High throughput quantitative proteomics may offer greater insight into the actual biology of the malignant cell than genome analysis and therefore, may be more useful in the development of personalized therapy. The objective of this study is to establish a proteomic signature predicting achievement of at least VGPR to initial treatment with bortezomib (Velcade®), pegylated liposomal doxorubicin, and dexamethasone (VDD). We previously reported preliminary proteomic profile of malignant plasma cells (PCs) obtained from a set of naïve MM pts enrolled in the VDD trial (Dytfeld et al., ASH 2009). Here we present the results of differential proteomic analysis of MM PCs of all available samples from the frontline VDD study (≥VGPR vs. <VGPR) using two independent and complementary quantitative proteomic platforms. We also compared the proteomic profile with gene expression data. Preliminary validation of the biomarkers of response prediction is presented. Methods: PCs were acquired from pre-treatment bone marrow specimens after obtaining informed consent from patients (pts), and were thereafter enriched with a RosetteSep® negative selection kit. Quantitative proteomic analysis of PCs from 17 naïve pts with MM from the VDD study was performed using iTRAQ approach in 8-plex variant. To increase confidence of analysis, label-free quantitative proteomics (LF) based on spectra counting was conducted on PCs from 12 pts. In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as a reference. The data were analyzed using ProteinPilot™. For LF analysis, proteins were fractionated before trypsin digestion on Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). A database search was carried out using X!Tandem followed by Trans-proteomic Pipeline. At least 1.5-fold difference in expression in both platforms was used as a cut-off value. To correlate proteomics with gene expression of dysregulated proteins of interest, mRNA levels were analyzed by quantitative real time PCR (RT-PCR). Validation of proteomic findings on proteins of interest was performed using Western Blot. Results: We identified a total of 894 proteins in 3 iTRAQ experiments with high confidence (FDR<1%) and 1058 proteins by LF approach. Based on iTRAQ analysis, 20 proteins were found up-regulated in samples from pts with ≥VGPR (8 out of 17 pts) while 14 were down- regulated. Using LF approach, 284 proteins were elevated in the ≥VGPR group (6 out of 12 pts) while 315 proteins were down-regulated. Both iTRAQ and LF methods showed 15 differentially expressed proteins in common and 14 of them showed identical up or down trends. Interestingly, among differentially expressed proteins, there were proteins involved in proteasome activation (PSME1 and TXNL1), protection against oxidative stress (TXN and TXNDC5), glucose and cholesterol metabolism (TP1, APOA1 and ACAT1) and apoptosis (MX1). RT-PCR performed on a subset of genes confirmed the trend in differential expression between pts with ≥VGPR and <VGPR for TXNDC5 and PSME1. No change in mRNA expression levels was observed in TXN, APOA1, TPI1 and MX1while the trend in expression was reversed for ACAT1. Western blot analysis performed to date validated differential expression of PSME1. Conclusions: We present patient-derived proteomic characteristics of MM cells using two independent proteomic platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline VDD study shows differential expression of 34 proteins in pts who achieved ≥VGPR vs. pts with <VGPR. Correlation with gene expression and further validation and functional analysis are in progress. This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Jakubowiak: Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

scholarly journals Proteomic analysis of differentially expressed proteins in normal human thyroid cells transfected with PPFP

2012 ◽  
Vol 19 (5) ◽  
pp. 681-694 ◽  
Author(s):  
Xinying Li ◽  
Zhiming Wang ◽  
Jianming Liu ◽  
Cane Tang ◽  
Chaojun Duan ◽  
...  
Keyword(s):  
Western Blot ◽  
Proteomic Analysis ◽  
Colony Formation ◽  
Fusion Gene ◽  
Differentially Expressed ◽  
Human Thyroid ◽  
Peroxisome Proliferator ◽  
Differentially Expressed Proteins ◽  
Thyroid Cells ◽  
Peroxisome Proliferator Activated Receptor

The fusion gene encoding the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) (designated as the PPFP gene) is oncogenic and implicated in the development of follicular thyroid carcinoma (FTC). The effects of PPFP transfection on the biological characteristics of Nthy-ori 3-1 cells were studied by MTT assay, colony formation, soft-agar colony formation, and scratch wound-healing assays as well as by flow cytometry. Furthermore, the differentially expressed proteins were analyzed on 2-DE maps and identified by MALDI-TOF-MS. Validation of five identified proteins (prohibitin, galectin-1, cytokeratin 8 (CK8), CK19, and HSP27) was determined by western blot analysis. PPFP not only significantly increased the viability, proliferation, and mobility of the Nthy-ori 3-1 cells but also markedly inhibited cellular apoptosis. Twenty-eight differentially expressed proteins were identified, among which 19 proteins were upregulated and nine proteins were downregulated in Nthy-ori 3-1PPFP(Nthy-ori 3-1 cells transfected with PPFP). The western blot results, which were consistent with the proteome analysis results, showed that prohibitin was downregulated, whereas galectin-1, CK8, CK19, and HSP27 were upregulated in Nthy-ori 3-1PPFP. Our results suggest that PPFP plays an important role in malignant thyroid transformation. Proteomic analysis of the differentially expressed proteins in PPFP-transfected cells provides important information for further study of the carcinogenic mechanism of PPFP in FTCs.

Proteomic profile of primary isolated rat mesangial cells in high-glucose culture condition and decreased expression of PSMA6 in renal cortex of diabetic ratsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 635-648 ◽  
Author(s):  
Zhiguo Li ◽  
Haojun Zhang ◽  
Xi Dong ◽  
Frank J. Burczynski ◽  
Patrick Choy ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Gel Electrophoresis ◽  
Renal Cortex ◽  
Mesangial Cells ◽  
Diabetic Rats ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Isolated Rat

Diabetic nephropathy (DN) is one of the most important complications of diabetic patients and is characterized histologically by an accumulation of extracellular matrix (ECM) protein in the glomerular mesangium. Therefore, mesangial cells likely play an important role in the development of diabetic nephropathy. Here, we employed proteomic techniques to investigate the protein profile of rat mesangial cells under high-glucose culture conditions. Primary isolated rat glomerular mesangial cells were cultured under different concentrations of glucose (5.4 mmol·L–1 for normal control and 30 mmol·L–1 for high glucose) for 0, 8, 16, and 72 h, as well as for 25 days. Cellular total proteins were isolated from these cells and employed for two-dimensional gel electrophoresis (2-DE). Differentially expressed proteins were identified by matrix-assisted laser desorption – ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of these proteins were documented in rat models of diabetes by Western blot. Rat mesangial cells were successfully isolated in the laboratory and their proliferation rates were significantly inhibited by high glucose. Two-dimensional gel electrophoresis analyses revealed 28 differentially expressed protein spots between the normal and high-glucose groups. After MALDI-TOF-MS analysis, all 28 protein spots were successfully identified with the peptide mass fingerprint (PMF) method. Representatively, SOD1, PCBP1 and PSMA6 were validated by Western blot analysis following protein extractions from the normal and high-glucose groups. Abundance of these proteins was consistent with that found in 2-DE. Moreover, expression of SOD1, PCBP1, and PSMA6 in renal cortex was further examined in two rat models of diabetes (streptozotocin-induced and spontaneous OLETF diabetic models). Abundance of SOD1 and PCBP1 proteins did not show any significant difference between normal control and diabetic rats. However, abundance of the PSMA6 protein was significantly reduced in the renal cortex of both STZ-induced and spontaneous OLETF diabetic rats. Proteomic analysis identified 28 differentially expressed proteins in primary isolated rat mesangial cells between normal and high glucose treatments. Expression of one identified protein was found to be consistent with expression in the renal cortex of two rat diabetic models. Therefore, identification of protein expression patterns in mesangial cells can be employed to develop a therapeutic target for treatment of diabetic nephropathy.

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