• Title/Summary/Keyword: 2D/MALDI-TOF

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Proteome Analysis of Paenibacillus polymyxa E681 Affected by Barley

  • Seul, Keyung-Jo;Park, Seung-Hwan;Ryu, Choong-Min;Lee, Yong-Hyun;Ghim, Sa-Youl
    • Journal of Microbiology and Biotechnology
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    • v.17 no.6
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    • pp.934-944
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    • 2007
  • Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the strain was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1,000 spots in the cell and 1,100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E68l cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG, spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-alanine ligase A, isopentenyl-diphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.

Identification of Differentially Expressed Proteins at Four Growing Stages in Chicken Liver

  • Lee, K.Y.;Jung, K.C.;Jang, B.G.;Choi, K.D.;Jeon, J.T.;Lee, J.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.10
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    • pp.1383-1388
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    • 2008
  • Because of high growth rate and large deposition of fat in the abdomen, the chicken has been used as a model organism for understanding lipid metabolism, fattening and growing. In this study, differentially expression of proteins in chicken liver, one of the important organs for lipid metabolism, has been investigated at four different growing stages. After separation of proteins using two-dimensional electrophoresis (2-DE), more than 700 protein spots were detected. Among them, 13 growing stage specific proteins in chicken liver were selected and further investigated by matrix-assisted laser adsorptions ionization-time of flight mass spectrometry (MALDI-TOF MS). Of these, 12 proteins were matched to existing proteins based on a database search. The identified fat-related proteins in this study were fatty acid synthase (FASN) and malic enzyme (ME1). These proteins were more highly expressed at week 32 than at other weeks. In order to confirm the differential expression, one of the proteins, FASN, was confirmed by western blotting. The identified proteins will give valuable information on biochemical roles in chicken liver, especially for lipid metabolism.

Determination of Stereochemical Structure of a Grandidentatin Isomer from Populus alba × glandulosa Bark (현사시나무 수피에서 분리한 Grandidentatin Isomer의 입체구조결정)

  • Kwon, Dong-Joo;Kim, Hyun-Seok;Lee, Phil-Ho;Bae, Young-Soo
    • Journal of the Korean Wood Science and Technology
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    • v.37 no.1
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    • pp.114-120
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    • 2009
  • The bark of Populus alba ${\times}$ glandulosa was collected, air-dried and extracted with 70% aqueous acetone. Then it was successively partitioned with n-hexane, $CH_2Cl_2$, EtOAc and $H_2O$. Repeated Sephadex LH-20 column chromatography and preparative TLC on the EtOAc soluble fraction gave a grandidentatin isomer. The structure was elucidated as grandidentatin A (cis-2-hydroxycyclohexyl 6-O-p-coumaroyl-${\beta}$-D-glucopyranoside) on the basis of spectroscopic evidences such as $^1H$-NMR, $^{13}C$-NMR, 2D-NMR and MALDI TOF-MS spectrum followed by acid hydrolysis. Grandidentatin A was identified here for the first time in Populus alba ${\times}$ glandulosa bark, and to the bset of our knowledge it has not been reported in any other literature.

Purification of Neuropeptide with the Contractile Activity on the Smooth Muscle from the Skin of Conger Eel Conger myriaster (붕장어(Conger myriaster)의 피부로부터 평활근 수축작용을 지닌 신경성 펩타이드의 정제)

  • Go, Hye-Jin;Park, Nam-Gyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.45 no.4
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    • pp.358-366
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    • 2012
  • A novel neuropeptide was isolated from the skin of the conger eel Conger myriaster using hagfish Eptatretus burgeri intestine as a bioassay system. The sequence of the purified peptide was analyzed using automated amino acid sequencing and MALDI-TOF mass spectrophotometry. The molecular ion peak in the MALDI-TOF mass spectrum of the peptide was at m/z 962.89 $(M+H)^+$. The sequence of the peptide was determined to be L-P-M-L-E-T-Q-M, and was tentatively named comyrin. To investigate the complete primary structure of comyrin, comyrin-OH and comyrin-$NH_2$ were synthesized and the chemical and pharmacological properties of the synthetic peptides were compared with those of the native peptide. However, the elution time of synthetic peptides did not match that of the native peptide on the reverse-phase HPLC chromatogram. In addition, the synthetic peptides did not cause contractile activity in the intestinal smooth muscle of the hagfish. Based on these results, one possible reason for this disagreement may be the presence of a D-amino acid in comyrin.

Differential protein expression in avian liver in response to invasion by Salmonella gallinarum

  • Lee, Gang-Deog;Cho, In-Hee;So, Hyun-Kyung;Koo, Yong-bum;Lee, Jun-heon;Choi, Kang-Duk
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 2004.11a
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    • pp.37-38
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    • 2004
  • Salmonella gallinarum is a pathogen that is capable of causing disease in Korean native chicken. Although Salmonella gallinarum is important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella gallinarum in the chicken. This study was to investigate chicken liver proteins affected by infection of Salmonella gallinarum in Korean native chicken. The differentially expressed proteins of chicken livers were identified by using 2-dimensional electro- phoresis (2D-E) and mass spectrometry (MS). We detected more than 300 protein spots on silver stained 2D gels using pH 3∼10 gradients. Three differentially expressed protein spots were analyzed by MALDI-TOF MS and MS/MS. The obtained MS and MS/MS data were searched against a protein database using the Mascot search engine. Further researches on the identified proteins can give valuable information of mechanism of pathogenesis in chicken.

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Proteomic Analysis of Global Changes in Protein Expression During Exposure of Gamma Radiation in Bacillus sp. HKG 112 Isolated from Saline Soil

  • Gupta, Anil Kumar;Pathak, Rajiv;Singh, Bharat;Gautam, Hemlata;Kumar, Ram;Kumar, Raj;Arora, Rajesh;Gautam, Hemant K.
    • Journal of Microbiology and Biotechnology
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    • v.21 no.6
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    • pp.574-581
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    • 2011
  • A Gram-positive bacterium was isolated from the saline soils of Jangpura (U.P.), India, and showed high-level of radiation-resistant property and survived upto 12.5 kGy dose of gamma radiation. The 16S rDNA sequence of this strain was examined, identified as Bacillus sp. strain HKG 112, and was submitted to the NCBI GenBank (Accession No. GQ925432). The mechanism of radiation resistance and gene level expression were examined by proteomic analysis of whole-cell extract. Two proteins, 38 kDa and 86.5 kDa excised from SDS-PAGE, which showed more significant changes after radiation exposure, were identified by MALDI-TOF as being flagellin and S-layer protein, respectively. Twenty selected 2-DE protein spots from the crude extracts of Bacillus sp. HKG 112, excised from 2- DE, were identified by liquid chromatography mass spectrometry (LC-MS) out of which 16 spots showed significant changes after radiation exposure and might be responsible for the radiation resistance property. Our results suggest that the different responses of some genes under radiation for the expression of radiation-dependent proteins could contribute to a physiological advantage and would be a significant initial step towards a fullsystem understanding of the radiation stress protection mechanisms of bacteria in different environments.

Muscle Proteome Analysis for the Effect of Panax Ginseng Extracts in Chicken: Identification of Proteins Using Peptide Mass Fingerprinting

  • Jung, K.C.;Yu, S.L.;Lee, Y.J.;Choi, K.D.;Choi, J.S.;Kim, Y.H.;Jang, B.G.;Kim, S.H.;Hahm, D.H.;Lee, J.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.7
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    • pp.922-926
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    • 2005
  • The present study was aimed to investigate proteome affected by Panax ginseng extracts in chicken muscles. The whole muscle proteins from chicken fed boiled extracts of 0% (control), 1%, 3%, and 5% Panax ginseng in water were separated by two-dimensional electrophoresis (2-DE) gels using immobilized non-linear gradient (pH 3-10) strips. More than 300 protein spots were detected on silver staining gels. Among them, four protein spots were distinctively up-regulated by Panax ginseng treatments and further investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The obtained MS data were searched against SwissProt database using the Mascot search engine. The up-regulated proteins were finally identified as $\alpha$-tropomyosin (2 spots), triosephosphate isomerase, and one unknown protein. Based on the known functions of the identified proteins, they are highly related to muscle development and enhanced immunity in chickens. These proteins can give valuable information of biochemical roles for Panax ginseng in chicken meats.

Bacterial Identification and Detection of Equol in Korean Soybean Paste (한국 된장에서 Equol의 검출 및 미생물 동정)

  • Woo, Seung-Gyun;Lee, So-Yeon;Choi, Go-Woon;Hong, You-Jin;Lee, So-Min;Park, Kang Gyun;Eom, Yong-Bin
    • Korean Journal of Clinical Laboratory Science
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    • v.47 no.4
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    • pp.286-291
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    • 2015
  • Equol has beneficial effects on human health. Fermented soy products contain equol, and many microbes participate in the equol production process. This study investigated fermented Korean soybean paste, doenjang. Thirty seven doenjang samples collected from different manufacturers were examined. Equol was detected in 3 samples (D2, D13, and D19) at the maximum content of 507 ng/100 g by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifteen microbial species were isolated and identified by 16S rRNA gene sequence analysis and by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Bacillus spp, Paenibacillus spp, Tetragenococcus spp, Stapylococcus spp, and Clostridium species were the predominant bacteria in equol containing doenjang samples.