• 제목/요약/키워드: 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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    • 제15권5호
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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.

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

  • 남원희;이선미;김은실;김진호;정진용
    • 생명과학회지
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    • 제17권5호
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    • pp.723-727
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    • 2007
  • 본 연구는 H. pylori에서 metronidazole내성에 관여하는 유전자를 발견하고 이들 유전자들의 상호 조절 기전을 밝힘으로서 위장질환의 원인균인 H. pylori를 퇴치하기 위한 기본바탕을 마련하고자 수행되었다. 우선적으로 metronidazole 내성을 조절하는 유전자인 fdxA(ferredoxin)에 의한 metronidazole 내성 조절 기전을 밝히기 위하여 다음의 연구를 수행하였다. Type I 균주인 26695균주의 fdxA 유전자에 chloramphenicol 내성 유전자를 삽입하여 결손돌연변이주를 구축하였다. fdxA의 비활성화에 의한 rdxA 및 frxA 유전자의 발현을 알아보기 위하여 2-D electrophoresis와 MALDI-TOP-MS을 이용하여 fdxA 유전자의 비활성화에 의해 over-expressed protein과 under-expressed protein을 검색하였다. 본 실험의 결과로 type I 균주인 26695에서 fdxA 유전자를 비활성화시킨 결과 frxA 유전자의 발현양이 증가함을 northern으로 확인하였으며, 또한 fdxA유전자의 downstream에 위치한 유전자들이 H. pylori의 생존에 중요한 역할은 한다는 것을 알 수 있었다. 또한 2-D electrophoresis와 MALDI-TOP-MS을 이용하여 fdxA 유전자의 inactivation에 의해 over-expressed protein으로 nifU-like protein(HP0221), frxA(HP0642), nonheme ferritin(HP0653)와 아직 기능이 밝혀지지 않은 hypothetical protein(HP0902) 등이 발견되었다. 그리고 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase(HP0089), (3R)-hydroxymyristoyl ACP dehydratase(HP1376)과 thioredoxin(HP1458)등이 under-expressed protein으로 발견되었다.

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

  • 유영삼;이현종;이택준;박헌
    • Journal of the Korean Wood Science and Technology
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    • 제37권1호
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    • pp.56-64
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    • 2009
  • 본 연구의 목적은 콩기름을 오존 산화 처리하여 기능기를 도입하고 이를 바탕으로 친환경 목재 접착제를 개발하는 것이다. 시판용 콩기름을 시간당 약 7.13 g의 오존을 발생시켜 15분, 30분, 60분, 120분간 오존산화 처리한 후 FT-IR, $^1H$-NMR, MALDI-TOF MS, GC/MS 측정하여 구조변화를 분석하였다. 기기분석 결과 오존산화처리에 의하여 콩기름의 불포화 이중결합이 개열되어 카르복실기 또는 알데하이드기가 생성됨을 확인하였다. 오존산화콩기름 변성 pMDI 접착제의 접착력은 오존처리 정도와 pMDI의 혼합비율에 따라 달라지며, KS F 3101 보통합판기준 내수 접착강도를 안정적으로 만족 시키는 조건은 오존산화 처리시간 30분~60분, pMDI와 1:1로 혼합한 경우이며, 준내수 접착강도를 만족시키는 범위는 오존산화 처리시간 30분~60분, pMDI의 혼합비 0.5이상, 상태접착강도를 만족시키는 범위는 오존산화 처리시간 15분~60분, pMDI의 혼합비 0.25 이상임을 알 수 있었다. 위의 실험결과를 종합하여 볼 때, 오존산화 처리한 식용유는 오존처리시간 및 pMDI의 혼합비율을 조절하면 상태접착력 뿐만 아니라 내수접착력이 요구되는 목질복합재료 제조용 접착제 제조가 가능함을 확인할 수 있었다.

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
    • 한국가금학회:학술대회논문집
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    • 한국가금학회 2005년도 제22차 정기총회 및 학술발표회
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    • pp.66-67
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    • 2005
  • 배자 생식세포 발달에 관련된 메카니즘을 밝혀내기 위해서, 닭 배자 생식기에서 추출한 원시 생식세포의 단백질체 지도를 만들었다. 총 500 배자를 6일간 배양하여 배자 생식기를 획득했고, 7-10일 배양 후, 배양된 원시생식세포는 2차원 젤 전기 영동법에 의해 분할되어 졌다. 유의적 발현 수준을 나타낸 많은 단백질 스팟 들은 MALDI-TOP 와 LC-MS/MS에 의해 확인되었으며, 89개의 단백질 스팟 중에 50개의 mass spectra 들이 데이터베이스에서 조류 단백질과 일치함을 확인하였다. 본 실험에서 행한 단백질체 지도는 형질전환 연구와 생식세포 생물학 분야에 중요한 참고 문헌으로 가치를 가질수 있을 것이다.

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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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    • 제37권2호
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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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    • 한국균학회 2015년도 춘계학술대회 및 임시총회
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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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