• Title/Summary/Keyword: MALDI-TOF Mass

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Interactome Analysis of Yeast Glutathione Peroxidase 3

  • Lee, Phil-Young;Bae, Kwang-Hee;Kho, Chang-Won;Kang, Sung-Hyun;Lee, Do-Hee;Cho, Sa-Yeon;Kang, Seong-Man;Lee, Sang-Chul;Park, Byoung-Chul;Park, Sung-Goo
    • Journal of Microbiology and Biotechnology
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    • v.18 no.8
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    • pp.1364-1367
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    • 2008
  • Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identity the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.

Isolation of a Novel Neuropeptide with Contractile Activity on the Smooth Muscle from the Snakehead Channa argus (가물치(Channa argus)로부터 평활근 수축활성 펩타이드의 정제)

  • Go, Hye-Jin;Park, Nam-Gyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.45 no.2
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    • pp.114-121
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    • 2012
  • A novel neuropeptide was isolated from the skin of the snakehead Channa argus using the dorsal retractor muscle (DRM) of a starfish Asterina pectinifera as a bioassay system. The amino acid sequence of the purified peptide was analyzed using automated sequencing and MALDI-TOF mass spectrophotometry. The primary structure of the purified peptide was determined to be Pro-Ala-Leu-Ala-Leu. To investigate the complete primary structure of this peptide, Pro- Ala-Leu-Ala-Leu-OH and Pro-Ala-Leu-Ala-Leu-NH2 were synthesized. The chemical and pharmacological properties of the synthetic peptides were compared with those of the native peptide. Both the native peptide and synthetic Pro-Ala- Leu-Ala-Leu-OH had identical behaviors on the reverse-phase and cation-exchange HPLC chromatograms. Synthetic Pro-Ala-Leu-Ala-Leu-OH showed contractile activity on the DRM, and the threshold concentration of this peptide was approximately $10^{-8}$ M. The maximal contractile effect ($E_{max}$) of this peptide was $294{\pm}45.4$% at $10^{-5}$ M.

Improvement of Permeability to Organic Solvent in Escherichia coli for a Toxicity Biosensor

  • Bae, Hee-Kyung;Shin, Pyong-Kyun;Song, Bang-Ho
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 2001.06b
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    • pp.14-16
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    • 2001
  • The outer membrane (OM) of gram-negative bacteria acts as an effective permeability barrier against noxious agents including several antibiotics and organic solvents, and lipopolysaccharide (LPS) is the key molecule for this function. Outer membrane modified mutants (Ml-166, M2-42, M3-21) of E. coli DH5$\alpha$/pBSl were selected through a mutation using EMS (ethyl-methane-sulfonate). Among the selected mutants, M3-21 was twice as sensitive as LumisTo $x^{ }$ to benzene and M2-41 was 8 times as sensitive as LumisTo $x^{ }$ to toluene. To identify the structural change in the membrane by mutation, the relative cell surface hydrophobicities and the absorption of the crystal violet to the organisms were measured. All the mutants absorbed more crystal violet than their parent and the absorption of crystal violet increased in cell walls as carbohydrate of lipopolysaccharide decreased. When the cell surface hydrophobicities of DH5/pBSl and its mutants were measured by the BATH, the hydrophobicities of mutants increased compared to their parent in several organic solvents. The difference of lipopolysaccharide between DH5/pBSl and its mutants was identified by various ways such as the SDS-PAGE gel, the screening of LPS molecular weights, the mass spectrometry, and MALDI-TOF.F.

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Proteomic Analysis of MCAo Ischemia Model Administered with Yukmijihwangtang (MCAo 허혈동물모델에서 육미지황탕 효능에 관한 프로테오믹스 연구)

  • Kim, Young-Ok;Cho, Dong-Wuk;Kang, Bong-Joo
    • Korean Journal of Oriental Medicine
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    • v.13 no.1 s.19
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    • pp.153-160
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    • 2007
  • In the post-genome era, analysis of the cellular transcriptome using microarray or the cellular proteome using a 2-D gel electrophoresis and MALDI-TOF mass spectrometry are most widely used. Stroke is one of the most important causes of death along with cancer and cardiac disease. When pathological change of cells in developed from cerebral ischemia accompanied by stroke administration of neuroprotective drugs before stroke can decreases the degeneration of neuronal cells. The purpose of the present study was to assess the neuroprotective effect and protein expression after administration of P004, middle cerebral artery model of cerebral ischemia in rats. SD rats were subjected to middle cerebral artery occlusion. P004 (1,000 mg/kg) was administered 2 times at 0, 90 minutes after middle cerebral artery occlusion (MCAo). Rats were killed at 48 hours, and infarct area and volume were determined by histology and computerized image analysis. We investigated the protein expression profile on the global ischemia induced by MCAo. This proteomic analysis enable us to identify several proteins differently expressed in infarct brain tissue. The aims of this study were to do investigation comparing the neuroprotection activities of P004 and to understand the mechanism of acted as neuroprotective drug.

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Synthesis and Characterization of Phenanthrene-substituted Fullerene Derivatives as Electron Acceptors for P3HT-based Polymer Solar Cells

  • Mi, Dongbo;Park, Jong Baek;Xu, Fei;Kim, Hee Un;Kim, Ji-Hoon;Hwang, Do-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.35 no.6
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    • pp.1647-1653
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    • 2014
  • 9,10-Bis(bromomethyl)phenanthrene reacted with fullerenes via a Diels-Alder reaction to give phenanthrene-substituted fullerene mono-adducts (PCMA) and bis-adducts (PCBA) as electron acceptors for organic photovoltaic cells (OPVs). The syntheses of the fullerene derivatives were confirmed by $^1H$ $^{13}C$ NMR spectroscopy and MALDI-TOF mass spectrometry. PCMA and PCBA showed better light absorption in the UV-visible region than $PC_{61}BM$. Their electrochemical properties were measured using cyclic voltammetry. Accordingly, the lowest unoccupied molecular orbital (LUMO) energy levels of PCMA and PCBA were -3.66 and -3.57 eV, respectively. Photovoltaic cells were fabricated with a ITO/PEDOT:PSS/poly(3-hexylthiophene)(P3HT):acceptor/LiF/Al configuration, where P3HT and PCBA are the electron donors and acceptors, respectively. The polymer solar cell fabricated using the P3HT:PCBA active layer showed a maximum power conversion efficiency of 0.71%.

Regulation of Protein Expression in Mouse Liver by Inorganic Arsenic: Proteomic Analysis (무기비소에 의한 마우스 간의 단백질 발현 조절 : 단백체 분석)

  • Jin Bo-Hwan;Seong Je-Kyung;Ryu Doug-Young
    • Environmental Mutagens and Carcinogens
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    • v.26 no.2
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    • pp.35-40
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    • 2006
  • Background: Inorganic arsenic is a human carcinogen that can target the liver, but its carcinogenic mechanisms are still unknown. Inorganic arsenic induces a spectrum of tumors including hepatocellular carcinoma in mice. Methods: Pregnant C3H mice were supplied with drinking water containing 50 ppm sodium arsenite during their pregnancy. The protein expression profile in the liver of 0.5-day-old. male offsprings exposed transplacentally to sodium arsenite was analyzed using protein 2D gel electrophoresis followed by mass spectrometry (MALDI-TOF). Results: Expression of proteins such as hydroxymethylglutaryl-CoA synthase mitochondrial precursor (HMG-CoA synthase), ${\beta}$-actin (cytoplasmic 1) and apolipoprotein A-IV precursor (Apo-AIV) were induced in mouse liver by sodium arsenite, while uricase (urate oxidase), guanine nucleotidebinding protein beta subunit 2-like 1 (RACK1) and fructose-bisphosphate aldolase B (Aldolase 2) were down-regulated. Summary: Expression of proteins that have been implicated in carcinogenesis, such as HMG-CoA, ${\beta}$-actin, and RACK1, was regulated in the liver of mice transplacentally exposed to inorganic arsenic.

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Molecular characterization of a lectin, BPL-4, from the marine green alga Bryopsis plumosa (Chlorophyta)

  • Han, Jong-Won;Yoon, Kang-Sup;Jung, Min-Gui;Chah, Kyong-Hwa;Kim, Gwang-Hoon
    • ALGAE
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    • v.27 no.1
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    • pp.55-62
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    • 2012
  • A novel lectin specific to N-acetyl-D-galactosamine as well as N-acetyl-D-glucosamine was isolated from Bryopsis plumosa and named as BPL-4. Sodium dodecyl sulfate polyacrylamide gel electrophorese (SDS-PAGE) and matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry data showed that this lectin was a monomeric protein with molecular weight 12.9 kDa. The N-terminal amino acid sequences of the lectin were determined by Edman degradation and the full cDNA sequence encoding this lectin was obtained using the degenerate primers designed from the amino acid sequence. The size of the cDNA was 414 bp containing single open reading frame (ORF) encoding the lectin precursor. The homology analysis showed that this lectin might belong to H lectin group. BPL-4 showed high sequence similarity (60.6%) to BPL-3, which is a previously reported lectin from the same species. The comparative analysis on the lectin's primary structure showed two conserved domains including one possible active domain of H lectin group.

Pituitary Adenoma Biomarkers Identified Using Proteomic Fingerprint Technology

  • Zhou, Kai-Yu;Jin, Hang-Huang;Bai, Zhi-Qiang;Liu, Chi-Bo
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.4093-4095
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    • 2012
  • Objective: To determine whether pituitary adenomas can be diagnosed by identifying protein biomarkers in the serum. Methods: We compared serum proteins from 65 pituitary adenoma patients and 90 healthy donors using proteomic fingerprint technology combining magnetic beads with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Results: A total of 42 M/Z peaks were identified as related to pituitary adenoma (P<0.01). A diagnostic model established based on three biomarkers (3382.0, 4601.9, 9191.2) showed that the sensitivity of diagnosing pituitary adenoma was 90.0% and the specificity was 88.3%. The model was further tested by blind analysis showing that the sensitivity was 88.0% and the specificity was 83.3%. Conclusions: These results suggest that proteomic fingerprint technology can be used to identify pituitary adenoma biomarkers and the model based on three biomarkers (3382.0, 4601.9, 9191.2) provides a powerful and reliable method for diagnosing pituitary adenoma.

Proteomic Approach to Study the Antioxidant Activities of Dioscoreae Rhizoma on HeLa Cells (산약(山藥)의 항산화 작용에 대한 단백질체 분석 연구)

  • Yang, Jeong-Min;Lee, Ji-Hyung;Sung, Jung-Suk;Kim, Dong-Il
    • The Journal of Korean Obstetrics and Gynecology
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    • v.21 no.2
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    • pp.108-124
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    • 2008
  • Purpose: This study was examined to verify the anti-oxidative effect of Dioscoreae Rhizoma on HeLa cells by proteomic approach. Methods: Aqueous extract was used to treat HeLa cell with different concentrations treated with water or MeOH extract of Dioscoreae Rhizoma. HeLa cells were co-treated with $H_2O_2$ and Dioscoreae Rhizoma extracts. Proteomics was done to identify, characterize, and quantitate proteins expressed in HeLa cells treated by $H_2O_2$ and Dioscoreae Rhizoma. Results: When HeLa cells were Co-treated with $H_2O_2$ and Dioscoreae batatas extracts, 16 proteins identified by 2-DE and MALDI-TOF mass spectrometry and database search. PRDX, HSP27 was major proteins of antioxidant effect by Dioscoreae batatas. Conclusion: Our results suggest that Dioscoreae Rhizoma extracts induce antioxidant effects by regulating proteins such as PRDX, HSP27.

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Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)

  • Kim, Young-Ha;Islam, Mohammad Saiful;You, Myung-Jo
    • Parasites, Hosts and Diseases
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    • v.53 no.1
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    • pp.85-93
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    • 2015
  • Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.