• Bulletin of the Korean Chemical Society
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• 제28권9호
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• pp.1499-1509
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• 2007

Proteomic analyses of synaptic vesicle fraction from rat brain have been performed for the better understanding of vesicle regulation and signal transmission. Two different approaches were applied to identify proteins in synaptic vesicle fraction. First, the isolated synaptic vesicle proteins were treated with trypsin, and the resulting peptides were analyzed using a high-pressure capillary reversed phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). Alternatively, proteins were separated by two-dimensional gel electrophoresis (2DE) and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS). Total 18 and 52 proteins were identified from cRPLC/MS-MS and 2DE-MALDI-TOF-MS analysis, respectively. Among them only 2 proteins were identified by both methods. Of the proteins identified, 70% were soluble proteins and 30% were membrane proteins. They were categorized by their functions in vesicle trafficking and biogenesis, energy metabolism, signal transduction, transport and unknown functions. Among them, 27 proteins were not previously reported as synaptic proteins. The cellular functions of unknown proteins were estimated from the analysis of domain structure, expression profile and predicted interaction partners.

• 한국작물학회지
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• 제60권1호
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• pp.29-32
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• 2015

Two-dimensional electrophoresis (2-DE) was executed to separate the seed storage proteins from the buckwheat. The proteins extracted from the whole seed proteins were better separated and observed in the use of lysis buffer. Using this method, the highly reproducible isoelectric focusing (IEF) can be obtained from polyacrylamide gels, and IEF from the polyacrylamide gel at all the possible pH range (5.0-8.0) was more easily separated than IPG (immobilized pH gradient) gels. The polyacrylamide gels in the first dimension in 2-DE was used to separate and identify a number of whole seed proteins in the proteome analysis. In this new apparatus using 2-DE, 27cm in length of plate coated with polyacrylamide gel was used and the experiment was further investigated under the various conditions.

• 생명과학회지
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• 제15권3호
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• pp.427-433
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• 2005

초파리는 유전학, 발달 생물학, 행동 유전학, 노화 연구에 이르기까지 수많은 연구에 이용 되어져 왔다. 최근 초파리 전체 유전자의 염기서열이 보고되었으며, 유전자의 기능 분석과 발현 단백질에 대한 연구가 진행되고 있다. 본 연구에서는 야생형 초파리에서 추출한 단백질을 이차원 전기영동을 통해 400여개 이상의 단백질 스폿으로 분리하였으며, 각 스폿을 적출하여 트립신으로 처리하여 얻어진 펩타이드 단편을 MALDI-TOF-MS를 이용한 펩타이드 지문 검색으로 질량을 측정하였다. 측정된 질량을 초파리 데이터 베이스를 이용하여 분리한 단백질을 동정함으로써 59개의 유전자에서 발현되는 65개의 단백질 스폿을 동정하였다. 이러한 결과는 향후의 발생단계, 외부 자극, 노화 등에 관련되는 특이적 단백질 연구의 기초 자료로 활용될 수 있을 것이다.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2002년도 제38회 학술심포지움
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• pp.20-47
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• 2002

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed In human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected on silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained by colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsln, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser dosorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, maps of lower resolution, i.e. overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.

• 대한미생물학회지
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• 제35권2호
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• pp.97-108
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• 2000

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• BMB Reports
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• 제40권6호
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• pp.853-860
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• 2007

Resistance to anticancer drugs is a major obstacle in the effective treatment of tumors. To understand the mechanisms responsible for multidrug resistance (MDR), a proteomic approach was used to identify proteins that were expressed in different levels by the adriamycinresistant human gastric cancer cell line, SGC7901/ADR, and its parental cell line, SGC7901. Two-dimensional gel electrophoresis (2-DE) and image analysis was used to determine which protein spots were expressed in different levels by the two cell lines. These spots were then partially identified using ESI-Q-TOF mass spectrometry, and the differential expressional levels of the partially identified proteins were then determined by western blot analysis and real-time RT-PCR. Additionally, the association of Nucleophosmin (NPM1), a protein that was highly expressed by SGC7901/ADR, with MDR was analyzed using siRNA. As a result of this study, well-resolved, reproducible 2-DE patterns of SGC7901/ADR and SGC7901 were established, and 16 proteins that may playa role in the development of thermo resistance were identified. Additionally, suppression of NPMl expression was found to enhance adriamycin chemosensitivity in SGC7901/ADR. These results provide a fundamental basis for the elucidation of the molecular mechanism of MDR, which may assist in the treatment of gastric cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.367-372
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• 2013

Effects of the Epstein-Barr virus (EBV) on cellular protein expression are essential for viral pathogenesis. To characterize the cellular response to EBV infection, differential proteomes of gastric epithelial AGS cells were analyzed with two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and liquid chromatography electrospray/ionization ion trap (LC-ESI-IT) mass spectrometry identification. Mass spectrometry identified 9 altered cellular proteins, including 5 up-regulated and 4 down-regulated proteins after EBV infection. Notably 2-DE analysis revealed that EBV infection induced increased expression of heat shock cognate 71 kDa protein, actin cytoplasmic 1, pyridoxine-5'-phosphate oxidase, caspase 9, and t-complex protein 1 subunit alpha. In addition, EBV infection considerably suppressed those cellular proteins of zinc finger protein 2, cyclin-dependent kinase 2, macrophage-capping protein, and growth/differentiation factor 11. Furthermore, the differential expressional levels of partial proteins (cyclin-dependent kinase 2 and caspase 9) were confirmed by Western blot analysis.Thus, this work effectively provided useful protein-related information to facilitate further investigation of the mechanisms underlying EBV infection and pathogenesis.

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1943-1950
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• 2016

Polycyclic aromatic hydrocarbons (PAHs) are commonly present xenobiotics in natural and contaminated soils. We studied three (phenanthrene, naphthalene, and biphenyl) xenobiotics, catabolism, and associated proteins in Sphingobium chungbukense DJ77 by two-dimensional gel electrophoresis (2-DE) analysis. Comparative analysis of the growth-dependent 2-DE results revealed that the intensity of 10 protein spots changed identically upon exposure to the three xenobiotics. Among the upregulated proteins, five protein spots, which were putative dehydrogenase, dioxygenase, and hydrolase and involved in the catabolic pathway of xenobiotic degradation, were induced. Identification of these major multifunctional proteins allowed us to map the multiple catabolic pathway for phenanthrene, naphthalene, and biphenyl degradation. A part of the initial diverse catabolism was converged into the catechol degradation branch. Detection of intermediates from 2,3-dihydroxy-biphenyl degradation to pyruvate and acetyl-CoA production by LC/MS analysis showed that ring-cleavage products of PAHs entered the tricarboxylic acid cycle, and were mineralized in S. chungbukense DJ77. These results suggest that S. chungbukense DJ77 completely degrades a broad range of PAHs via a multiple catabolic pathway.

• 환경생물
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• 제22권4호
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• pp.487-493
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• 2004

To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.