• Title/Summary/Keyword: LC-MALDI

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Comparative Proteome Analysis of Cyanidin 3-O-glucoside Treated Helicobacter pylori

  • Kim, Sa-Hyun;Kim, Jong-Bae
    • Biomedical Science Letters
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    • v.21 no.4
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    • pp.233-240
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    • 2015
  • Some virulence proteins of Helicobacter pylori, such as vacuolating cytotoxic protein A (VacA) and cytotoxin-associated gene protein A (CagA) have been reported to be causative agents of various gastric diseases including chronic gastritis, gastric ulcer or gastric adenocarcinoma. The expression level of these virulence proteins can be regulated when H. pylori is exposed to the antibacterial agent, cyanidin 3-O-glucoside (C3G) as previously reported. In this study, we analyzed the quantitative change of various virulence proteins including CagA and VacA by C3G treatment. We used 2-dimensional electrophoresis (2-DE) to analyze the quantitative change of representative ten proteome components of H. pylori 60190 ($VacA^+/CagA^+$; standard strain of Eastern type). After 2-DE analysis, spot intensities were analyzed using ImageMaster$^{TM}$ 2-DE Platinum software then each spot was identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using Finnigan LCQ ion trap mass spectrometer (LC-MS/MS). Next, we selected major virulence proteins of H. pylori among quantitatively meaningful ten spots and confirmed the 2-DE results by Western blot analysis. These results suggest that cyanidin 3-O-glucoside can modulate a variety of H. pylori pathogenic determinants.

Difference in Susceptibility of Tyrosine Residue to Oxidative Iodination between a Thioredoxin Box Region and a Hormonogenic Region

  • Sok, Dai-Eun;Charles J.Sih
    • Archives of Pharmacal Research
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    • v.24 no.5
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    • pp.446-454
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    • 2001
  • Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

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The Effect of Protein Expression of Streptococcus pneumoniae by Blood

  • Bae, Song-Mee;Yeon, Sun-Mi;Kim, Tong-Soo;Lee, Kwang-Jun
    • BMB Reports
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    • v.39 no.6
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    • pp.703-708
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    • 2006
  • During infection, the common respiratory tract pathogen Streptococcus pneumoniae encounters several environmental conditions, such as upper respiratory tract, lung tissue, and blood stream, etc. In this study, we examined the effects of blood on S. pneumoniae protein expression using a combination of highly sensitive 2-dimensional electrophoresis (DE) and MALDI-TOF MS and/or LC/ESI-MS/MS. A comparison of expression profiles between the growth in THY medium and THY supplemented with blood allowed us to identify 7 spots, which increased or decreased two times or more compared with the control group: tyrosyl-tRNA synthetase, lactate oxidase, glutamyl-aminopeptidase, L-lactate dehydrogenase, cysteine synthase, ribose-phosphate pyrophosphokinase, and orotate phosphoribosyltransferase. This global approach can provide a better understanding of S. pneumoniae adaptation to its human host and a clue for its pathogenicity.

Proteomics Analysis of Gastric Epithelial AGS Cells Infected with Epstein-Barr Virus

  • Ding, Yong;Li, Xiao-Rong;Yang, Kai-Yan;Huang, Li-Hua;Hu, Gui;Gao, Kai
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.1
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    • pp.367-372
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    • 2013
  • Effects of the Epstein-Barr virus (EBV) on cellular protein expression are essential for viral pathogenesis. To characterize the cellular response to EBV infection, differential proteomes of gastric epithelial AGS cells were analyzed with two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and liquid chromatography electrospray/ionization ion trap (LC-ESI-IT) mass spectrometry identification. Mass spectrometry identified 9 altered cellular proteins, including 5 up-regulated and 4 down-regulated proteins after EBV infection. Notably 2-DE analysis revealed that EBV infection induced increased expression of heat shock cognate 71 kDa protein, actin cytoplasmic 1, pyridoxine-5'-phosphate oxidase, caspase 9, and t-complex protein 1 subunit alpha. In addition, EBV infection considerably suppressed those cellular proteins of zinc finger protein 2, cyclin-dependent kinase 2, macrophage-capping protein, and growth/differentiation factor 11. Furthermore, the differential expressional levels of partial proteins (cyclin-dependent kinase 2 and caspase 9) were confirmed by Western blot analysis.Thus, this work effectively provided useful protein-related information to facilitate further investigation of the mechanisms underlying EBV infection and pathogenesis.

Proteomics Analysis of Early Salt-Responsive Proteins in Ginseng (Panax ginseng C. A. Meyer) Leaves (초기 염류 스트레스 반응 인삼 잎 단백질체 분석)

  • Kim, So Wun;Min, Chul Woo;Gupta, Ravi;Jo, Ick Hyun;Bang, Kyong Hwan;Kim, Young-Chang;Kim, Kee-Hong;Kim, Sun Tae
    • Korean Journal of Medicinal Crop Science
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    • v.22 no.5
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    • pp.398-404
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    • 2014
  • Salt stress is one of the major abiotic stresses affecting the yield of ginseng (Panax ginseng C. A. Meyer). The objective of this study was to identify bio-marker, which is early responsive in salt stress in ginseng, using proteomics approach. Ginseng plants were exposed to 5 ds/m salt concentration and samples were harvested at 0, 6, 12 and 18 hours after exposure. Total proteins were extracted from ginseng leaves treated with salt stress using Mg/NP-40 buffer and were separated on high resolution 2-DE. Approximately $1003{\pm}240$ (0 h), $992{\pm}166$ (6 h), $1051{\pm}51$ (12 h) and $990{\pm}160$ (18 h) spots were detected in colloidal CBB stained 2D maps. Among these, 8 spots were differentially expressed and were identified by using MALDI-TOF/TOF MS or/and LC-MS/MS. Ethylene response sensor-1 (spot GL 1), nucleotide binding protein (spot GL 2), carbonic anhydrase-1 (spot GL 3), thylakoid lumenal 17.9 kDa protein (spot GL 4) and Chlorophyll a/b binding protein (spot GL 5, GL 6) were up-regulated at the 12 and 18 hour, while RuBisCO activase B (spot GL 7) and DNA helicase (spot GL 8) were down-regulated. Thus, we suggest that these proteins might participate in the early response to salt stress in ginseng leaves.

Proteomic Analysis of Protein Expression in Streptococcus pneumoniae in Response to Temperature Shift

  • Lee Myoung-Ro;Bae Song-Mee;Kim Tong-Soo;Lee Kwang-Jun
    • Journal of Microbiology
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    • v.44 no.4
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    • pp.375-382
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    • 2006
  • From its initial colonization to causation of disease, Streptococcus pneumoniae has evolved strategies to cope with a number of stressful in vivo environmental conditions. In order to analyze a global view of this organism's response to heat shock, we established a 2-D electrophoresis proteome map of the S. pneumoniae D39 soluble proteins under in vitro culture conditions and performed the comparative proteome analysis to a 37 to $42^{\circ}C$ temperature up-shift in S. pneumoniae. When the temperature of an exponentially growing S. pneumoniae D39 culture was raised to $42^{\circ}C$, the expression level of 25 proteins showed changes when compared to the control. Among these 25 proteins, 12 were identified by MALDI-TOF and LC-coupled ESI MS/MS. The identified proteins were shown to be involved in the general stress response, energy metabolism, nucleotide biosynthesis pathways, and purine metabolism. These results provide clues for understanding the mechanism of adaptation to heat shock by S. pneumoniae and may facilitate the assessment of a possible role for these proteins in the physiology and pathogenesis of this pathogen.

Proteomics of plant-fungal pathogen interaction: an overview (식물과 곰팡이 병원균과의 상호작용에 대한 프로테오믹스 최근 연구 동향)

  • Kim, Jin Yeong;Lee, So Eui;Oh, Ha Ram;Choi, In Soo;Kim, Yong Chul;Kim, Sun Tae
    • Journal of Plant Biotechnology
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    • v.41 no.1
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    • pp.1-9
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    • 2014
  • So far it has been generally considered that proteomic approaches are very useful for studying plant-microbes interaction. In this review, recent studies based on papers published from 2010 to 2013 have investigated proteomics analysis in various interaction during plant-fungal pathogen infection by means of gel-based proteomics coupled with mass spectrometry (MS)-based analysis. In rice, three papers focused on rice-Magnaporthe oryzae interaction were mainly reviewed in this study. Interestingly, another study showed proteomic changes in rice inoculated with Puccinia triticina, which is not only an fungal pathogen in wheat and but also results to the disease resistance with non-host defense manner in rice. Additionally, proteomics analysis has been widely subjected to understand defense mechanism during other crops (wheat, tomato, strawberry and mint) and their fungal pathogen interaction. Crops inoculated are analyzed to identify differentially regulated proteins at various tissues such as leaf and apoplast using 2-DE analysis coupled with various MS approaches such as MALDI-TOF MS, nESI-LC-MS/MS and MudPIT, respectively. Taken together, this review article shows that proteomics is applicable to various organisms to understand plant-fungal pathogen interaction and will contribute to provide important information for crop disease diagnosis and crop protection.

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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