• Title/Summary/Keyword: MALDI-TOF/TOF MS

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Toxicoproteomics in the Study of Aromatic Hydrocarbon Toxicity

  • Cho, Chang-Won;Kim, Chan-Wha
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.3
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    • pp.187-198
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    • 2006
  • The aromatic hydrocarbons (AHs), which include benzene, polycyclic aromatic hydrocarbons, and dioxin, are important chemical and environmental contaminants in industry that usually cause various diseases. Over the years, numerous studies have described and evaluated the adverse health effects induced by AHs. Currently, "Omics" technologies, transcriptomics and proteomics, have been applied in AH toxicity studies. Proteomics has been used to identify molecular mechanisms and biomarkers associated with global chemical toxicity. It could enhance our ability to characterize chemical-induced toxicities and to identify noninvasive biomarkers. The proteomic approach (e.g. 2-dimensional electrophoresis [2-DE]), can be used to observe changes in protein expression during chemical exposure with high sensitivity and specificity. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization-quadrupole (ESI-Q)-TOF MS/MS are recognized as the most important protein identification tools. This review describes proteomic technologies and their application in the proteomic analysis of AH toxicity.

Identification and Molecular Characterization of Three Isoforms of Iturin Produced by Endophytic Bacillus sp. CY22 (식물 내생균 Bacillus sp. CY22가 생성하는 iturin isoform의 분리 및 특성)

  • Cho, Soo-Jeong;Yun-Han-Dae
    • Journal of Life Science
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    • v.15 no.6 s.73
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    • pp.1005-1012
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    • 2005
  • Endophytic Bacillus sp. CY22 was previously isolated from the interior of balloon flower root and showed strong antifungal activity against phytopathogenic fungi such as Rhizoctonia solnni, Fusarium oxysporum, and Phythium ultimum. Many Bacillus strains produce antifungal compound such as iturin, fengycin, and mycosubtilin. We isolated and identified antifungal compound from cell supernatant of the endophytic strain. By the MALDI-TOF mass result, the antifungal compound was similar to the known antifungal lipopeptide iturin. It was found that the purified iturin had three isoforms with protonated masses of m/z 1,043.39, 1,057.42, and 1,071.42 and different structures in combination with $Na^{+}$ ion using MALDI-TOF MS. The ita22 gene, which transacylase gene is associated with production of antifungal iturin, had an open reading frame (ORF) of 1,200 bp encoding 400 amino acids. Results of deduced amino acids sequence homology search, Ita22 was homologous with FenF (BAB69697) of Bacillus subtilis 168.

Application of Malononitrile Derivatization Method for Structural Glycomics Study in Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

  • Ahn, Yeong-Hee;Yoo, Jong-Shin
    • Journal of Photoscience
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    • v.8 no.2
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    • pp.83-86
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    • 2001
  • Structural analyses of oligosaccharide-malononitrile derivatives were conducted by matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) analysis in positive ion mode. The malononitrile derivatives of oligosaccharides, which were developed for highly sensitive detection of multi-component oligosaccharides by negative ion electrospray ionization mass spectrometry (ESI MS), were detected by positive-ion MALDI with the detection limit of 2 pmol level from the crude derivatization sample. The used matrix affected drastically the analytical results of oligosaccharide-malononitrile derivative by matrix-assisted laser desoprtion/ionization mass spectrometry (MALDI MS). The malononitrile derivatization of oligosaccharide also affect the patterns of MALDI-PSD spectra and give much more structural information than the free oligosaccharide.

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Increased Viability of Sub-lethal Heat Shocked Salmonella Typhimurium on Acids and Oxidants (열충격 Salmonella Typhimurium의 산과 산화제에서 생존력 증가)

  • Moon, Bo-Youn;Park, Jong-Hyun
    • Korean Journal of Food Science and Technology
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    • v.40 no.6
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    • pp.712-716
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    • 2008
  • In an effort to evaluate Salmonella food safety using combinations of preservation techniques, its viabilities when exposed to HCl, acetic acid, and the oxidative agents (hydrogen peroxide and butyl hydrogen peroxide), were analyzed using sub-lethal heat-shocked Salmonella Typhimurium at $56^{\circ}C$. 2D gel electrophoresis and MALDI-TOF MS analyses were also conducted to determine the expression and repression of proteins in heat-shocked cells. Heat-shocked S. Typhimurium evidenced a reduction of viable counts by 1-2 log CFU/mL. However, viality of non heat-shocked S. Typhimurium decreased markedly by 5-6 log CFU/mL at a pH 4 in response to acid and oxidative stresses. Sub-lethal heat treatment greatly increased the resistance of S. Typhimurium against acid and oxidant agents. As for 2D gel electrophoresis and protein identification via MALDI-TOF MS, 17 major proteins in non heat-shocked S. Typhimurium were detected, and only 13 proteins among these proteins were detected in heat-shocked S. Typhimurium. The heat shock proteins such as DnaK and small heat shock proteins were included, and may be associated with the resistance of S. typhimurium against exposure to acids and oxidants. Therefore, even though the promising hurdle technology using the combined mild treatments including heat was applied to S. Typhimurium, the proper heat treatment to reduce its crossprotection activity toward the following preservative agents might be considered.

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.

Identification of LAB and Fungi in Laru, a Fermentation Starter, by PCR-DGGE, SDS-PAGE, and MALDI-TOF MS

  • Ahmadsah, Lenny S.F.;Kim, Eiseul;Jung, Youn-Sik;Kim, Hae-Yeong
    • Journal of Microbiology and Biotechnology
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    • v.28 no.1
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    • pp.32-39
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    • 2018
  • Samples of Laru (a fermentation starter) obtained from the upper part of Borneo Island were analyzed for their lactic acid bacteria (LAB) and fungal diversity using both a culture-independent method (PCR-DGGE) and culture-dependent methods (SDS-PAGE and MALDI-TOF MS). Pediococcus pentosaceus, Lactobacillus brevis, Saccharomycopsis fibuligera, Hyphopichia burtonii, and Kodamaea ohmeri were detected by all three methods. In addition, Weissella cibaria, Weissella paramesenteroides, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Rhizopus oryzae/Amylomyces rouxii, Mucor indicus, and Candida intermedia were detected by PCR-DGGE. In contrast, Lactobacillus fermentum, Lactobacillus plantarum, Pichia anomala, Candida parapsilosis, and Candida orthopsilosis were detected only by the culture-dependent methods. Our results indicate that the culture-independent method can be used to determine whether multiple laru samples originated from the same manufacturing region; however, using the culture-independent and the two culture-dependent approaches in combination provides a more comprehensive overview of the laru microbiota.

Purification and Characterization of PC-Like Cadmium-Binding Peptide from Root of Rumex crispus

  • Chang, Ju-Youn;Lee, In-Sook;Park, Jin-Sung;Chang, Yoon-Young;Bae, Bum-Han
    • The Korean Journal of Ecology
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    • v.26 no.5
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    • pp.263-266
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    • 2003
  • This research investigated the process of removing cadmium and tested the detoxification mechanism of the cadmium-binding peptide (Cd-BP) from Rumex crispus. Phytochelatin-like cadmium-binding peptide (PC-Cd-BP) of Rumex crispus was purified and identified. Rumex crispus was exposed to 4.3 mg Cd/L for seven days. Heat-treated supernatant fraction taken by root tissues showed traces of PC-Cd-BP An analysis of the material through Gel-filteration chromatography on the Sephadex G-75 column showed two symmetrical Cd-BP peaks. The major peak with the smaller molecular weight was further purified by $C_{18}$ reverse-phase HPLC to produce apparent homogeneity. The amino acid composition of Cd-BP from Rumex crispus included cysteine (22.6%), glutamate and glutamate acid (20%), and glycine (12%). It was similar the amino acid composition of most PC. The molecular weight of the purified peptide was determined at 568-706 Da by MALDI-TOF MS. Therefore, the Cd-BP of Rumex crispus was PC-Cd-BP consisting of isopeptides.

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.

Purification and Properties of Chitosanase from Chitinolytic $\beta$-Proteobacterium KNU3

  • Yi, Jae-Hyoung;Jang, Hong-Ki;Lee, Sang-Jae;Lee, Keun-Eok;Choi, Shin-Geon
    • Journal of Microbiology and Biotechnology
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    • v.14 no.2
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    • pp.337-343
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    • 2004
  • A bacterial strain concurrently producing extracellular chitosanase and chitinase was isolated from soil and identified as a member of the $\beta$-subgroup of Proteobacteria through its 16S rRNA analysis and some biochemical analyses. The newly discovered strain, named as KNU3, had 99% homology of its 16S rRNA sequence with chitinolytic $\beta$-Proteobacterium CTE108. Strain KNU3 produced 34 kDa of chitosanase in addition to two chitinases of 68 kDa and 30 kDa, respectively. The purified chitosanase protein (ChoK) showed activity toward soluble, colloidal, and glycol chitosan, but did not exhibit any activity toward colloidal chitin. The optimum pH and temperature of ChoK were 6.0 and $70^{\circ}C$, respectively. The chitosanase was stable in the pH 4.0 to 8.0 range at $70^{\circ}C$, while enzyme activity was relatively stable at below $45^{\circ}C$. MALDI-TOF MS and N-terminal amino acid sequence analyses indicated that ChoK protein is related to chitosanases from Matsuebacter sp. and Sphingobacterium multivorum. HPLC analysis of chitosan lysates revealed that glucosamine tetramers and hexamers were the major products of hydrolysis.