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

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Purification and Characterization of Manganese Peroxidase of the White-Rot Fungus Irpex lacteus

  • Shin Kwang-Soo;Kim Young Hwan;Lim Jong-Soon
    • Journal of Microbiology
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    • v.43 no.6
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    • pp.503-509
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    • 2005
  • The production of manganese peroxidase (MnP) by Irpex lacteus, purified to electrophoretic homogeneity by acetone precipitation, HiPrep Q and HiPrep Sephacryl S-200 chromatography, was shown to correlate with the decolorization of textile industry wastewater. The MnP was purified 11.0-fold, with an overall yield of $24.3\%$. The molecular mass of the native enzyme, as determined by gel filtration chromatography, was about 53 kDa. The enzyme was shown to have a molecular mass of 53.2 and 38.3 kDa on SDS-PAGE and MALDI-TOF mass spectrometry, respectively, and an isoelectric point of about 3.7. The enzyme was optimally active at pH 6.0 and between 30 and $40^{\circ}C$. The enzyme efficiently catalyzed the decolorization of various artificial dyes and oxidized Mn (II) to Mn (III) in the presence of $H_2O_2$. The absorption spectrum of the enzyme exhibited maxima at 407, 500, and 640 nm. The amino acid sequence of the three tryptic peptides was analyzed by ESI Q- TOF MS/MS spectrometry, and showed low similarity to those of the extracellular peroxidases of other white-rot basidiomycetes.

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

  • Song, Hyun-Ouk
    • Parasites, Hosts and Diseases
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    • v.54 no.1
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    • pp.71-74
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    • 2016
  • Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Changes of Protein Profiles in Cheonggukjang during the Fermentation Period (전통 청국장의 발효 기간 동안 변화하는 수용성 단백질 개요)

  • Santos, Ilyn;Sohn, Il-Young;Choi, Hyun-Soo;Park, Sun-Min;Ryu, Sung-Hee;Kwon, Dae-Young;Park, Cheon-Seok;Kim, Jeong-Hwan;Kim, Jong-Sang;Lim, Jin-Kyu
    • Korean Journal of Food Science and Technology
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    • v.39 no.4
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    • pp.438-446
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    • 2007
  • The fermented soybean product, cheonggukjang, is favored by many people, partly due to its bio-functional ingredients. Since the fermentation process of cheonggukjang is mediated by enzymes, including proteases, produced by microbes, analysis of the proteome profile changes in cheonggukjang during fermentation would provide us with valuable information for fermentation optimization, as well as a better understanding of the formation mechanisms of the bio-functional substances. The soluble proteins from cheonggukjang were prepared by a phenol/chloroform extraction method, in order to remove interfering molecules for high resolution 2-D gel analysis. Proteomic analysis of the cheonggukjang different fermentation periods suggested that most of the soluble soy proteins were degraded into smaller forms within 20hr, and many microbial proteins, such as mucilage proteins, dominated the soluble protein fraction. The proteomic profile of cheonggukjang was very different from natto, in terms of the 2-D gel protein profile. Among the separated protein spots on the 2-D gels, 50 proteins from each gel were analyzed by MALDI-TOF MS and PMF for protein identification. Due to database limitations with regard to soy proteins and microbial proteins, identification of the changed proteins during fermentation was restricted to 9 proteins for cheonggukjang and 15 for natto. From de novo sequencing of the proteins by a tandem MS/MS, as well as by database searches using BLASTP, a limited number of proteins were identified with low reliability. However, the 2-D gel analysis of proteins, including protein preparation methods, remains a valuable tool to analyze complex mixtures of proteins entirely. Also, for intensive mass spectrometric analysis, it is also advisable to focus on a few of the interestingly changed proteins in cheonggukjang.

복제 소 태반과 IVF 소 태반의 protein pattern 분석

  • 김홍래;강재구;윤종택;성한우;조민래;박창식;진동일
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.90-90
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    • 2003
  • 체세포 핵이식에 의한 복제기술은 매우 낮은 성공률 나타내고 있어 실용화에 지장을 초래하고 있다. 이것은 후생적인 유전현상인 reprogramming이 불완전하게 이루어지기 때문인 것으로 추측되어지고 있다(Reik et al., Theriogenology 2003, 59: 21-32; Han et al, Theriogenology 2003, 59: 33-44). 체세포 핵이식 후에 태아사망의 원인이 태반의 비정상적인 기능과 관계가 있는 것으로 추정되는데 복제시 태아사망의 원인을 찾기 위해 본 연구를 시행하였다. 한우에서 체세포 복제 후 임신 말기에 태아가 사망한 태반조직 3개와 IVF 수정란 이식 후 동일한 시기에 제왕절개술을 실시한 태반조직 2개를 실험에 이용하였다. 태반 protein을 Two-Dimensional electrophoresis와 Mass spectrometer를 이용하여 분석 비교하였다. IPG-system을 이용하여 pH 4~7, pH 6~9에서 1차 전기영동을 한 후, 8~l6%의 SDS-PAGE gel에 2차 전기영동을 실시하였고 G-250 Coomassie로 염색하였다. gel 이미지는 Malanie III program을 이용하여 분석하였다. 전체 gel에서 약 1800개의 구분 가능한 protein spot이 나타났다. pH 4~7 범위에서 양적으로 차이나는 것 15개 중 복제한우 태반에서 증가되는 protein spot 5개와 감소하는 protein spot 10개를 골라 protein identification을 실시하였다. MALDI-TOF-MS를 이용하여 동정한 결과 phosphatidylinositol transfer protein-$\alpha$와 interleukin-18 등의 protein이 복제태반에서 발현이 증가되었고, 복제한우에서 발현이 감소되는 것으로는 vimentin, Rho-GDI-$\beta$, TRAST $\beta$-chain, ovarian sterol carrier protein 2, triosephosphate isomerase, tropemyosin beta chain, Aldose reductase 등으로 나타났다. 이러한 protein들은 inositol 지질 신호전달과 면역시스템, 세포분열, 산소 운반, steroidogenic 세포에서의 콜레스테롤 이동, 촉매 작용, 대사 작용 등에 중요한 역할을 하는 것으로 알려져 체세포 복제에 의한 태아사망 원인은 태반에서 이러한 protein들의 비정상적인 발현에 기인된 것으로 추정된다.

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Characterization of a Fibrinolytic Metalloenzyme from a Wild Mushroom, Tricholoma sejunctum (쓴송이버섯으로부터 분리한 혈전용해 금속효소의 특성 연구)

  • Kim, Jun-Ho;Cho, Seung-Koo
    • The Korean Journal of Mycology
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    • v.32 no.2
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    • pp.119-124
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    • 2004
  • Metalloenzyme was purified from the fruiting bodies of Tricholoma sejunctum. MALDI-TOF and ICP/MS analyses revealed that the enzyme had a molecular weight of 18788.25 and includes $Zn^{2+}$ ion. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. The activity of the enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme was a metalloprotease. No inhibition was found with E-64 and pepstatin. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $40^{\circ}C$. The activity of the enzyme was increased by $Zn^{2+}$ and $Co^{2+}$, while it was totally inhibited by $Hg^{2+}$. The enzyme hydrolyzes $A{\alpha}$ subunit of human fibrinogen but did not show any reactivity for $B{\beta}$ and ${\gamma}$ form of human fibrinogen.

Characteristics of Wood Tar Produced as Byproduct from Two Types of The Kiln in The Manufacture of Oak Charcoal

  • Yang, Bong Suk;Yang, Jiwook;Kim, Dae-Young;Kim, Jin-Kyu;Hwang, Won-Jung;Kwon, Gu-Joong
    • Journal of the Korean Wood Science and Technology
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    • v.45 no.6
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    • pp.772-786
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    • 2017
  • This study investigated the characteristics of wood tar, produced as a byproduct during the production of charcoal using oak wood by the modified traditional kiln and mechanical steel kiln. The wood tar was analyzed with a number of techniques, including Py-GC/MS, NMR, MALDI-TOF, FT-IR, TG and DSC. The Py-GC/MS analysis indicated that modified traditional kiln generated a higher hydrocarbon ratio in the wood tar than that of mechanical steel kiln. On the other hand, mechanical steel kiln resulted in a higher proportion of phenolic and aromatic hydrocarbon components than that of modified traditional kiln. Those results were also confirmed by NMR analysis. The MALDI-TOF analysis suggested that the wood tar produced in the mechanical steel kiln had a slightly higher molecular weight than the wood tar produced in the modified traditional kiln. In addition, the FT-IR analysis showed characteristic peak of symmetrical stretching vibration of $CH_3$ from the modified traditional kiln while characteristic peaks of the C-C and C-O stretching vibration were observed from the mechanical steel kiln. Moreover, TG and DSC analysis suggested that the mechanical steel kiln is more thermally stable than that of modified traditional kiln. Those findings clearly showed that the method of making charcoal greatly affects the properties of wood tar.