Expression of Raf kinase inhibitor protein in human hepatoma tissues by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption/ionization time‑of‑flight methods.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor‑specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. MATERIALS AND METHODS: In this study, we use two‑dimensional polyacrylamide gel electrophoresis (2‑DE) and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. RESULTS: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf‑mediated activation of mitogen‑activated protein kinase/extracellular signal‑regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time‑polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non‑neoplastic liver tissue of the corresponding HCC samples. CONCLUSION: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down‑regulated in liver cancer cell.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective Under the methodology of differential proteomics and bioinformatics,the impact of the exogenous gene on the expression of rice in the proteome is discussed,aiming to explore into the study of genetically modified rice in the proteomics. Methods The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63 (MH63),the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional poly?acrylamide gel(2D-PAGE)electrophoresis spectrum;then,the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results The comparing and matching of protein spots between transgenic Bt (cry1Ab/1Ac)rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-trans?genic rice as a reference,transgenic Bt rice held 18 relatively high and 10 relatively low expressions;mass spectrometry and bioinfor?matics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy me?tabolism,protein synthesis,redox stress response and other biological processes. Conclusion Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome;however,there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective: Under the methodology of differential proteomics and bioinformatics, the impact of the exogenous gene on the expression of rice in the proteome is discussed, aiming to explore into the study of genetically modified rice in the proteomics. Methods: The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63(MH63), the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional polyacrylamide gel(2D-PAGE) electrophoresis spectrum; then, the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results: The comparing and matching of protein spots between transgenic Bt(cry1Ab/1Ac) rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-transgenic rice as a reference, transgenic Bt rice held 18 relatively high and 10 relatively low expressions; mass spectrometry and bioinformatics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy metabolism, protein synthesis, redox stress response and other biological processes. Conclusion: Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome; however, there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Candidates for Tumor Markers of Cervical Cancer Discovered by Proteomic Analysis

Cervical cancer is the second most common gynecological cancer among Korean women. While nationwide screening program has developed, the pathogenesis of cervical cancer is unknown. The aim of this study was to compare the protein expression profiles between cervical squamous carcinomas and normal cervical tissues in order to identify proteins that are related to the cancer. Three cervical cancer tissue samples and three normal cervical tissue samples were obtained and protein expression was compared and was identified in the samples with the use of matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). A total of 20 proteins that showed up-regulated expression in the cervical cancer tissue samples were selected and identified. Seven proteins were matched to allograft inflammatory factor 1 (AIF-1), actine-like protein 2 (ALP2), brain type fatty acid-binding protein (B-FABP), NCK adaptor protein 1 (NCK-1), islet cell autoantigen 1 (ICA69), cationic trypsinogen (PRSS1), and cyclin-dependent kinase 4 (CDK4), but the remaining 13 proteins were unidentifiable. After confirmation by RT-PCR, Western blotting and immunohistochemistry, we found that B-FABP, NCK-1, and CDK4 were related to the pathogenesis of cervical cancer. These proteins are suggested as candidates of new pathological tumor markers for cervical cancer.

Proteome Analysis of Hirschsprung Disease by Two-dimensional Electrophoresis / 中国医科大学学报

Objective To study the protein expression profiles in stegnotic and normal segment of Hirschspning disease (HD) and find the differentially expressed proteins. Methods Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to isolate the proteins from stegnotic and normal segment of HD. After the samples were treated with silver staining,ImageMaster 2D Platinum analysis software was used to analyze the differentially expressed proteins. Results Repeatable 2-DE profiles were obtained. The mean matching rates of the stegnotic and normal mucosa were 78.1% and 86.7%,respectively. Totally, 103 spots of differentially expressed proteins were screened out between the stegnotic and the normal segment of HD. Conclusion Good reproduuibility and resolution could be obtained in the tissues of HD by applying immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis.Screened differentially expressed proteins may provide the candidates of the markers for early detection of HD.

Selection and identification of differential proteins in plasma/serum with hepatocellular carcinoma by proteomic techniques / 重庆医科大学学报

Objective:To establish a two-dimensional polyacrylamide gel electrophoresi(s2-DE)technique forplasma/serumproteomics. Methods:Plasmas from 8 healthy volunteers and 10 with hepatocellular carcinoma(HCC),and sera from 8 HCC were collected.After six proteins of the highest abundance in plasma/sera were depleted by the Multiple Affinity Removal System(MARS)Technologies from Agilent,plasma/sera were subjected to 2-DE.After comparing,analyzing and identifying by the software PD Quest(7.4,0),the enzyme-digested products of differential expression proteins were analyzed by MALDI-TOF-MS.The Peptide mass fingerprinting(PMF) data were obtained and used to identify proteins through NCBInr data bank.Results:In comparison with the healthy volunteers,20 differential proteins were identified in the plasmas of the HCC patients,among which 18 protein expressions were up-regulated and 2 down-regulated.In the sera of the cancer patients,23 differential proteins were identified,among which 17 protein expressions were up-regulated and 6 down-regulated.Tumor necrosis factor-inducible protein TSG-14 and FADD protein were both identified in the plasmas and sera of the cancer patients.Conclusion:The differential proteins,including RING finger protein 34,Wilms’tumor 1-associating protein,CD147 antigen,Tumor necrosis factor-inducible protein TSG-14 from plasmas group and Protein kinase NYD-SP9,Tumor necrosis factor receptor super family member 19 precursor,Histone-binding protein RBBP4,FADD protein,26S protease regulatory subunit p28,CD82 antigen,Autophagy protein 5 from sera group may be used as markers for HCC in blood.

Selection and Optimization of 2-DE System for Leaf Proteome Profiling of Different Ecotypes of Reed Growing in Natural Habitats / 生物化学与生物物理进展

An optimized two-dimensional polyacrylamide gel electrophoresis (2-DE) system for analyzing plant proteins was developed by evaluating different reagents and concentrations used in the sample extraction solutions and lysis buffers. Two main sample preparation methods, referred to as trichloroacetic acid (TCA)-acetone method and phenol extraction-ammonium acetate/methanol (phenol-NH4Ac/methanol) precipitation method, were compared. Four ecotypes of reed plants (Phragmites communis Trin.) from the desert region of north-western China were used as experimental materials: (1) swamp reed (SR) which grows in water about 1 m deep; (2) dune reed (DR) which grows on 5～10 m high sand dunes; (3) heavy salt meadow reed (HSMR) which grows on low-lying salt flats; and (4) light salt meadow reed (LSMR) which grows in the transition area between DR and HSMR growing areas. The optimized phenol-NH4Ac/methanol precipitation method consisted of extracting leaf proteins of different ecotypes of reed with water-saturated phenol and then precipitating with a 5-fold volume of 0.1 mol/L NH4Ac in methanol, followed by dissolving in the lysis buffer. The optimized protein lysis buffer consisted of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 2% Ampholine(pH 3.5～10∶pH 5～8 = 1∶4) and 65 mmol/L DTT. The prepared protein sample (80 ?g) was then separated by 2-DE gel and detected by silver staining method. This improved 2-DE system resulted in a 2-D protein profile of higher resolution and higher protein yields as analyzed by PDQuest software. Good results were also obtained when this 2-DE system was used in 2-D analysis of proteins from other plant materials, such as rice leaves, indicating that it is a suitable 2-DE system for analyzing leaf proteins of different plant species.

Differential Proteomics of Outer Membrane Proteins Between Virulent and Avirulent Strains of Riemerella anatipestifer / 生物化学与生物物理进展

In order to identify virulence factors of the pathogen, the outer membrane proteins of virulent and avirulent strains of Riemerella anatipestifer were compared by a proteome analysis. Three protein spots differentially expressed between the two strains were observed by 2-DE gels, and were further analyzed using in gel tryptic digestion and peptide mass fingerprinting. Three proteins were identified. W1 was Hsp20, W2 and W3 were transposon. Although the exact role of these proteins has not been characterized, the exclusive expression in virulent strain may indicate that they play an important role in the pathogenesis of Riemerella anatipestifer infection. Although only two virulence factors identified, it opens a path to the further analysis of virulence factors of Riemerella anatipestifer.

Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. MATERIALS AND METHODS: Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software(TM). The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. RESULTS: A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, alpha-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. CONCLUSION: 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC.

Establishment of two-dimensional polyacrylamide gel electrophoresis for serum proteomics / 重庆医科大学学报

Objective: To establish a two-dimensional polyacrylamide gel electrophoresis(2-DE) technique for serum proteomics.Methods: Various conditions of serum proteomic 2-DE were adjusted and optimized.Results: A steady 2-DE technique was established.Conclusions: Our established 2-DE technique of serum proteins can be effectively applied in serum proteomics of diseases.

Comparative proteomic analysis of differently differentiated human laryngeal squamous cell cancer / 第三军医大学学报

Objective To establish two-dimensional electrophoresis profiles of laryngeal squamous carcinoma tissue with high resolution and reproducibility. Methods The total protein of well-differentiated or poorly-differentiated laryngeal cancer tissue was separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differential expression protein was analyzed by using ImageMaster 2D analysis software. Results We obtained fine resolution, reproducible 2-DE patterns of well-differentiated and poorly-differentiated laryngeal squamous carcinoma. Differential expression protein spots were defined in 2-DE gels. Thirteen protein spots were preliminarily identified, among which seven proteins were upregulated in well-differentiated laryngeal cancer tissue, six were downregulated. Conclusion The expression map of proteomics was established for well-differentiated and poorly-differentiated laryngeal cancer, which was of significant difference between them. Laryngeal cancer proteome might reflect the underlying pathological state.

Specific plasma protein detected on physical education athletic students / 体力科学

The purpose of this study is to analyze the plasma proteins of physical education athletic and general students (aged 19-22) before their breakfast by two-dimensional electrophoresis.<BR>The results of this study, a protein which has not been found in any original report yet is detcted from physical education athletic students before breakfast.<BR>This protein was not detected from general studenets before breakfast. This protein was detected at the position of pI5.0, molecular weight of about 70, 000 on two-dimensional polyacrylamide gel electrophosis under non-denaturing codition, but it showed a molecular weight of about 30, 000 on SDS-polyacrylamide gel electrophoresis.<BR>As far as this research is concerned, neither special physical education athletic students and nor general students were observed in changes of two-dimensional electrophoretic patterns of blood cell cytosol and blood cell membrane proteins.