• Journal of Applied Biological Chemistry
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• 제51권3호
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• pp.88-94
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• 2008

Three different protein extraction methods-phenol extraction, trichloroacetic acid (TCA) precipitation, and desalting/TCA precipitation-were compared to determine the optimal reproducible high resolution 2-dimensional (2-D) electrophoresis for each chungkugjang, doenjang, and kochujang samples. The soluble proteins from Chungkugjang extracted by phenol were separated with high reproducibility and resolution, and gained 1.75- to 3-fold more protein spots on 2-D gel than those from the other methods. On the contrary, the extracted proteins from doenjang and kochujang treated by desalting/TCA precipitation method showed about 1.5- to 3.3-fold more protein spots on 2-D gel. Using the established methods, the changes in the protein profiles of the fermented soy foods were monitored during the fermentation period by 2-DE. One of the major proteins in soy, $\beta$-conglycinin $\alpha$-subuint, and some proteins with unknown functions were localized on 2-D gel as the protease-resistant proteins throughout the fermentation period of doenjang. Changes in the protein profile monitored by the established methods can provide basic information on unfolding the mechanisms of the generation of biofunctional activity in the fermented soy foods.

• 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
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• 제44권4호
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• pp.369-374
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• 2006

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis $D-8^T$. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain $D-8^T$ are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and $Imagemaster^{TM}$ 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, S proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.

• Journal of Plant Biology
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• 제36권4호
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• pp.377-382
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• 1993

벼(Oryza sativa L.) 캘러스는 2.0 mg/L 2,4-D와 0.5 mg/L kinetin이 첨가된 MS 배지에서 성숙종자로부터 유도되었으며 embryogenic callus(EC)와 nonembryogenic callus(NEC)는 색깔과 외부형태에 의해 경시적으로 선별되었다. EC와 NEC의 전체 단백질로부터 SDS-PAGE와 등전점 전기영동에 의한 전기영동적 분석은 EC와 NEC의 각각에 대해 특이적, 양적인 차이를 보여주었다. 또한 EC와 NEC의 2차원 전기영동 분석은 약 20여개 이상의 EC 특이단백질과 10여개의 NEC 특이 단백질 양상을 보여주었으며, 아울러 EC 특이적인 90, 65, 50 kD의 단백질은 microheterogeneity를 보여주는 반면, NEC에서는 분자량의 변이가 큰 일련의 산성 이질단백질군을 보여주었다.

• Journal of Plant Biotechnology
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• 제37권2호
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• pp.196-204
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• 2010

With the completion of genome sequencing of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. The recent techniques of proteomics have been advanced quickly so that the high-throughput and systematic analyses of cellular proteins are enabled in combination with bioinformatics tools. Furthermore, the development of proteomic techniques helps to elucidate the functions of proteins under stress or diseased condition, resulting in the discovery of biomarkers responsible for the biological stimuli. Ultimate goal of proteomics orients toward the entire proteome of life, subcellular localization, biochemical activities, and their regulation. Comprehensive analysis strategies of proteomics can be classified as three categories: (i) protein separation by 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification by either Edman sequencing or mass spectrometry (MS), and (iii) quanitation of proteome. Currently MS-based proteomics turns shiftly from qualitative proteome analysis by 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, to quantitative proteome analysis. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. The in vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes, protein-labeling tagging with isotope-coded affinity tag, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope labeled amino acid can be in vivo labeled into live culture cells through metabolic incorporation. MS-based proteomics extends to detect the phosphopeptide mapping of biologically crucial protein known as one of post-translational modification. These complementary proteomic techniques contribute to not only the understanding of basic biological function but also the application to the applied sciences for industry.

• 대한수의학회지
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• 제44권1호
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• pp.57-64
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• 2004

The common pathogen Salmonella enteirtidis (S. enteritidis) is the major cause of foodborne disease. Protein identification by peptide mass fingerprinting using the matrix-assisted laser desorption ionization time of fight (MALDI-TOF) mass spectrometry (MS) can analysis unambiguously identity the spots from 2-dimensional electrophoresis (2-DE) gel. In this report, we examined protein components from patterns of S. enteritidis proteins. In addition, antigens that are recognized by sera can be identified by immunoblotting. This study that 2-DE analysis of S. enteritidis yields useful information concerning S. enteritidis proteome, the results that have been obtained led to a more detailed understanding of Salmonella pathology and open further interesting fields for future work.

• 한국수정란이식학회지
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• 제23권1호
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• pp.25-30
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• 2008

This study was performed to identify and analyze the specifically expressed plasma proteins during early pregnancy in both pregnant and non-pregnant Hanwoo. Blood samples were collected at 0 (the day of AI), 2, 3, 4, 7, and 11 weeks after AI from pregnant (n=3) and non-pregnant (n=4) Hanwoo, respectively. The hematological parameters were measured. After 2-dimensional electrophoresis using serum, normalized protein spots were selected for the significant expression variation deviated over two fold in its expression level between two groups. Among 17 spots selected, 15 were identified as albumin, IgG1 heavy chain constant region, haptoglobin, ferrochelatase, fibrinogen, hemopexin. 5 spots were expressed only in non-pregnant specific. The spot identification of 1105 and 6106 was decreased after 3 weeks from AI. However, 2/17 spots were still unidentified. Further studies are needed to analyze the function of the proteins associated with early pregnancy.

• 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.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.173-175
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• 2004

단백질체(Proteome)이란 말은 어원적으로 단백질(protein)에 전체란 뜻을 가진 어미(-body, -some)가 연결된 합성어로 주어진 순간에 세포나 조직이 발현하는 모든 단백질의 총체를 의미하고 이를 연구하는 학문은 Proteomics라 일컫는다. 2001년 2월 International Human Genome Project에 의해 human genome sequence가 밝혀짐으로써 유전체 연구는 일단락 완성되었지만, 염기서열만 가지고는 이 유전자 산물의 기능을 알 수 없었고, 이것이 전사되고 최종적으로 완벽한 모양이 갖추어진 단백질을 분석해야만 그 기능을 알 수 있었다. (중략)

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.147.2-147.2
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• 2003

A fast and matrix-assisted laser desorption/ionization-mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, Zincon and Ethyl Violet to form an ion-pair complex. It is safe to use since the methanol used previously in staining solution was replaced with ethanol, which is not toxic. The protocol including fixing, staining and quick washing steps can be completed in 1 to 1.5 h depending upon gel thickness. (omitted)