• Title/Summary/Keyword: MALDI-TOP MS

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Mass-Spectral Identification of an Extracellular Protease from Bacillus subtilis KCCM 10257, a Producer of Antibacterial Peptide Subtilein

  • SONG HYUK-HWAN;GIL MI-JUNG;LEE CHAN
    • Journal of Microbiology and Biotechnology
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    • v.15 no.5
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    • pp.1054-1059
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    • 2005
  • An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

Mechanism of Metronidazole Resistance Regulated by the fdxA Gene in Helicobacter pylori. (헬리코박터 파일로리에서 fdxA 유전자에 의한 메트로니다졸 내성 조절 기전 연구)

  • Nam, Won-Hee;Lee, Sun-Mi;Kim, Eun-Sil;Kim, Jin-Ho;Jeong, Jin-Yong
    • Journal of Life Science
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    • v.17 no.5 s.85
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    • pp.723-727
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    • 2007
  • Resistance to metronidazole in Helicobacter pylori results from inactivation of rdxA and frxA, the chromosomal genes for a nitroreductase that normally converts metronidazole from prodrug to bactericidal agent. Two types of metronidazole susceptible strains had been found distinguishable by their apparent levels of frxA expression. Most common in the populations we had studied were strains that required only rdxA inactivation to become resistant to moderate levels of metronidazole(type I strains). The second strain type required inactivation of both frxA and rdxA to become resistance to metronidazole(type II strains): this was linked to a relatively high level of frxA gene transcription in the type II strains. The fdxA gene regulated fdxA as well as rdxA gene. Thus, to study the function of fdxA as a regulatory gene we constructed a null mutant of fdxA in H. pylori genome and identified over-and under-expressed proteins by fdxA using two-dimensional(2-D) electrophoresis and MALDI-TOP-MS. There were four over-expressed proteins in fdxA mutant; nifU-like protein(HP0221), frxA(HP0642), nonheme ferritin(HP0653), and hypothetical protein(HP0902). Three under-expressed proteins were also identified in fdxA mutant, including 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (HP0089), (3R)-hydroxymyristoyl ACP dehydratase(HP1376), and thioredoxin(HP1458).

Studies on Chemical Strutures and Adhesion Performance of pMDI Adhesives Modified by Ozonized Soybean Oil with Different Mixing Ratios (오존산화 콩기름의 구조분석 및 이를 이용한 변성 pMDI 접착제의 중량비에 따른 접착력 변화)

  • You, Young Sam;Lee, Hyun Jong;Lee, Taek Jun;Park, Heon
    • Journal of the Korean Wood Science and Technology
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    • v.37 no.1
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    • pp.56-64
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    • 2009
  • The purpose of this study was to investigate and develop an eco-friendly wood adhesive based on vegetable oil (especially soybean oil), the renewable and sustainable natural resources, using ozonification technology for the chemical structure modification. The soybean oil (SBO) was reacted with $O_3$ at the rate of 7.13 g/h for different times, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. The investigation of the modified chemical structure of the ozonized SBOs were conducted using FT-IR, $^1H$-NMR, MALDI-TOF MS, and GC/MS. As ozonification time increased, the peak of the unsaturated double bonds was disappeared and aldehyde or carboxyl peak appeared because ozonification broke the oil into small molecules. The plywoods were made at $110^{\circ}C$ with 30 seconds/mm hot-press time using the different ozonized SBO/pMDI adhesives and were tested for the dry, wet, cyclic boil test according to the Korea Industrial Standard F3101 Ordinary plywood. The bond strengths gradually increased with increasing ozonification time. The weight ratio 1:1 (ozonized SBO/pMDI), all strengths in 15, 30 and 60 minuets, exceeded constantly the dry, wet, cyclic boiling standard requirement. The range of ozonification time and weight ratio can fulfil1 the requirment of the wet test standard were 30~60 minutes and more than 0.5 pMDI. From the comprehensive view on the results of above experiments, it could be confirmed through experiments that ozonized SBO/pMDI has characteristics of effective reactivity and wet stability showed as an excellent candidate of wood adhesive applications.

Proteome Analysis of Chicken Embryonic Gonads: Identification of Major Proteins from Cultured Gonadal Primordial Germ Cells

  • Lee, Sang-In;Han, Beom-Ku;Park, Sang-Hyun;Kim, Tae-Min;Sin, Sang-Soo;Lee, Young-Mok;Kim, Hee-Bal;Lim, Jeong-Mook;Han, Jae-Yong
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 2005.11a
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    • pp.66-67
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    • 2005
  • The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.

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Comprehensive proteome analysis using quantitative proteomic technologies

  • Kamal, Abu Hena Mostafa;Choi, Jong-Soon;Cho, Yong-Gu;Kim, Hong-Sig;Song, Beom-Heon;Lee, Chul-Won;Woo, Sun-Hee
    • Journal of Plant Biotechnology
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    • v.37 no.2
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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.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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