liquid chromatography tandem mass spectrometry Search Results


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  • 99
    Thermo Fisher easy nlc 1000
    Easy Nlc 1000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tmz
    HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM <t>VPA,</t> 10 μ M SAHA or 100 μ M <t>TMZ.</t> Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM GSH. Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P
    Tmz, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies 1100 hplc system
    ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography <t>(HPLC)</t> on an Agilent <t>1100</t> HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.
    1100 Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 8933 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1200 hplc system
    The catumaxomab sample was analyzed using <t>RP-HPLC</t> mass spectrometry using a PLRP-S column (Varian) with an acetonitrile gradient from 27% to 42% over 40 minutes on a <t>1200</t> HPLC system (Agilent) for separation, coupled with an Agilent 6220 ESI-TOF mass
    1200 Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 8192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ultimate 3000 hplc system
    High resolution mass spectra of apo- and holo-LhaS. Spectra of apo-LhaS isolated from E. coli grown in RPMI medium (upper panel), holo-LhaS isolated from LB-medium (middle panel) and a heme standard (lower panel) were recorded on a HPLC-UV-HR mass spectrometer. The samples were diluted with MilliQ-H 2 O and applied to a Dionex <t>Ultimate</t> 3000 HPLC system that is coupled to the MaXis 4G ESI-QTOF mass spectrometer.
    Ultimate 3000 Hplc System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies gas chromatography mass spectrometry gc ms
    T. pseudethanolicus furan aldehyde reduction. T. pseudethanolicus was grown at 65°C with 40 mM glucose and 15 mM (A) furfural or (B) 5-HMF. Growth was measured by optical density at 600 nm. Furan aldehyde concentration was measured spectrophotometrically, while furan alcohol concentration was measured by <t>gas</t> <t>chromatography-mass</t> <t>spectrometry.</t> Error bars are the standard deviation of three replicate cultures.
    Gas Chromatography Mass Spectrometry Gc Ms, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 1718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher q exactive mass spectrometer
    The Q Exactive Hybrid <t>Quadrupole-Orbitrap-MS</t> spectrum of compound S in positive mode.
    Q Exactive Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher q exactive orbitrap mass spectrometer
    Quantitative affinity purification mass spectrometry for comparing NvHsp70 isoform interactions under heat stress. (A) NvHsp70 isoforms were expressed with an N-terminal 6xHIS tag in yeast lacking native Hsp70s. After exposing cells to heat stress (39 °C for 2 h), chaperone interactomes were isolated by nickel-NTA magnetic bead affinity purification. Following PAGE and in-gel trypsin digestion, peptides were analyzed by <t>Orbitrap</t> LC–MS/MS and MassQuant informatic analysis, allowing identification of interacting proteins and determination of their relative enrichment after heat stress. Each experiment was performed in biological triplicate. (B) Expression of NvHsp70 isoforms in yeast. Lysate of cells treated as in (A) were analyzed by SDS-PAGE/Western Blotting using pan-Hsp70 and GAPDH antibodies.
    Q Exactive Orbitrap Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies hplc system
    Trans -acylation reaction assays of various AT4 FkbB mutants after substitution of Val187 with Asp, or Cys, Ile, Lys, Trp, and Phe with allmal- and ethmal-CoA as substrates and the Production of <t>FK506</t> and FK520 in WT, YN06-01, and YN06-02. (A) The change of transferring allmal/ethmal unit to ACP10 FkbA in trans -acylation reactions by AT4 FkbB and its mutants V187D, V187C, V187I, V187K, V187W, and V187F. <t>HPLC</t> analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (B) and in the absence of AT4 FkbB (D) . MS analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (C) and in the absence of AT4 FkbB (E) . The peaks were assigned as follows: b 0 , holo-ACP10 FkbA ; b 1 , allmal-ACP10 FkbA ; b 2 , ethmal-ACP10 FkbA . (F) Production of FK506 and FK520 in WT, YN06-01 and YN06-02. Analyses of variance were conducted to determine the difference of WT and mutants using SPSS 20. The LSD multiple range tests were evaluated for significant differences among WT and mutants ( P
    Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 8784 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TriLink o6 methyl dgtp
    MTH1 is an efficient catalyst of <t>O6-methyl-dGTP</t> hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
    O6 Methyl Dgtp, supplied by TriLink, used in various techniques. Bioz Stars score: 99/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ltq linear ion trap mass spectrometer
    Representative standard curves of the Hepcidin-25H calibration. (A) A linear response of Hepcidin-25H quantitation between 0.5 to 6 picomoles was demonstrated in <t>TSQ</t> Quantum Ultra, (B) The linear response of the Hepcidin-25H quantification between 100 to 500 femtomoles was demonstrated in <t>LTQ-XL</t> linear ion trap instrument.
    Ltq Linear Ion Trap Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher orbitrap fusion mass spectrometer
    Metabolic Labeling and Workflow. ( A ) Glucose, acetate, fatty acids, and amino acids produce acetyl-CoA for use in acetylating cytoplasmic and nuclear proteins. The thicker arrows indicate that glucose contributes more to the production of acetyl-coA that subsequently acetylates proteins, compared to acetate. ( B ) The workflow consisted of growing HeLa cells in heavy-labeled media, collecting samples at eight time points, lysing the cells, digesting the proteins, enriching for acetylated peptides, and analyzing the peptides by mass spectrometry. The <t>Orbitrap</t> image is adapted from Thermo Fisher Scientific 56 . The cartoon cell matter and lab equipment were slightly modified from Servier Medical Art 57 .
    Orbitrap Fusion Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies esi source
    <t>HPLC-electrospray</t> <t>(ESI)-negative</t> mass spectrum of metabolites of SKN dependent on NADPH, UDPGA, or both cofactors. Mass spectrum of SKN ( A ), mass spectrum of M1 ( B ), mass spectrum of M2 ( C ), mass spectrum of M3 ( D ), mass spectrum of M4 ( E ), mass spectrum of M5 ( F ), and mass spectrum of M6 ( G ) are shown. The metabolites are identified in Table 3 .
    Esi Source, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nonlinear Dynamics progenesis lc ms software
    Estimated power versus putative type 1 error rate (α) with validation <t>(Progenesis</t> PPA measurements). a , power results for Progenesis PPA measurement data (as in ). At α = 0.05, the estimated power of ProPCA is 0.60, the estimated
    Progenesis Lc Ms Software, supplied by Nonlinear Dynamics, used in various techniques. Bioz Stars score: 91/100, based on 653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shimadzu Corporation shimadzu hplc system
    Estimated power versus putative type 1 error rate (α) with validation <t>(Progenesis</t> PPA measurements). a , power results for Progenesis PPA measurement data (as in ). At α = 0.05, the estimated power of ProPCA is 0.60, the estimated
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    Thermo Fisher ultimate 3000 hplc
    Estimated power versus putative type 1 error rate (α) with validation <t>(Progenesis</t> PPA measurements). a , power results for Progenesis PPA measurement data (as in ). At α = 0.05, the estimated power of ProPCA is 0.60, the estimated
    Ultimate 3000 Hplc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ. Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM GSH. Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P

    Journal: Cell Death & Disease

    Article Title: Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe

    doi: 10.1038/cddis.2014.412

    Figure Lengend Snippet: HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ. Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM GSH. Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P

    Article Snippet: Temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo-[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, TMZ), valproic acid (VPA), SAHA (suberanilohydroxamic acid, vorinostat), L- reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Immunofluorescence, Viability Assay, Flow Cytometry, Cytometry

    SAHA and VPA reduce the levels of expression of G2 checkpoint gatekeepers Wee1 and Chk1. ( a ) Analysis of the effect of HDACi on Wee1 and Chk1 protein expression and on the phosphorylation status of cdc2 (Tyr-15) in glioma cells. Cell cycle regulators, Cyclin B1, p21 and cdc25A, were also analyzed. Cells were treated for 24 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ, and protein extracts were analyzed by electrophoresis and western blot using specific antibodies against proteins indicated on the left of the panels. Representative blots of three independent experiments are shown. ( b ) Analysis of Wee1 and Chk1 mRNA after 24 h of HDACi treatment of glioma cell lines by reverse transcription and quantitative real-time PCR. Bars depict the mean±S.E.M. of four independent experiments. ( c ) Expression of Mus81 endonuclease in glioma cells treated as described above by western blot. Equal loading was verified by α -tubulin detection on the same membrane. ( d ) Downregulation of the endonuclease Mus81 do not prevent DSBs formation in SAHA-treated U251-MG cells. The efficacy of two specific shRNA against the mRNA of Mus81 (1 and 2) was verified by western blot (upper panel). Percentage of γH2AX-positive cells after 48 h of incubation in the presence of 10 μ M SAHA or 100 μ M TMZ of lentivirus-transduced cells. Data are mean±S.E.M. from three independent experiments. ( e ) Analysis of DNA fragmentation after Mus81 downregulation with shRNA. Representative gels of three independent experiments are shown, of high-molecular weight DNA fragmentation (upper panel) and low-molecular weight DNA fragmentation (lower panel) analysis

    Journal: Cell Death & Disease

    Article Title: Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe

    doi: 10.1038/cddis.2014.412

    Figure Lengend Snippet: SAHA and VPA reduce the levels of expression of G2 checkpoint gatekeepers Wee1 and Chk1. ( a ) Analysis of the effect of HDACi on Wee1 and Chk1 protein expression and on the phosphorylation status of cdc2 (Tyr-15) in glioma cells. Cell cycle regulators, Cyclin B1, p21 and cdc25A, were also analyzed. Cells were treated for 24 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ, and protein extracts were analyzed by electrophoresis and western blot using specific antibodies against proteins indicated on the left of the panels. Representative blots of three independent experiments are shown. ( b ) Analysis of Wee1 and Chk1 mRNA after 24 h of HDACi treatment of glioma cell lines by reverse transcription and quantitative real-time PCR. Bars depict the mean±S.E.M. of four independent experiments. ( c ) Expression of Mus81 endonuclease in glioma cells treated as described above by western blot. Equal loading was verified by α -tubulin detection on the same membrane. ( d ) Downregulation of the endonuclease Mus81 do not prevent DSBs formation in SAHA-treated U251-MG cells. The efficacy of two specific shRNA against the mRNA of Mus81 (1 and 2) was verified by western blot (upper panel). Percentage of γH2AX-positive cells after 48 h of incubation in the presence of 10 μ M SAHA or 100 μ M TMZ of lentivirus-transduced cells. Data are mean±S.E.M. from three independent experiments. ( e ) Analysis of DNA fragmentation after Mus81 downregulation with shRNA. Representative gels of three independent experiments are shown, of high-molecular weight DNA fragmentation (upper panel) and low-molecular weight DNA fragmentation (lower panel) analysis

    Article Snippet: Temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo-[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, TMZ), valproic acid (VPA), SAHA (suberanilohydroxamic acid, vorinostat), L- reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Electrophoresis, Western Blot, Real-time Polymerase Chain Reaction, shRNA, Incubation, Molecular Weight

    Pharmacological inhibition of Wnt/β-catenin signalling augments the cytotoxic effects of temozolomide. ( a ) Cell viability of cancer cell lines treated with increasing concentrations of different Wnt inhibitors and temozolomide (TMZ). Cell growth was assessed by FMCA or WST-1 after 72 h. All drugs were combined with a fixed molar ratio: TMZ:Celecoxib, 33:1; TMZ:G007-LK, 50:1; TMZ:LGK974, 20:1; TMZ:Wnt-C59, 20:1; TMZ:Salinomycin, 40:1; and TMZ:XAV-939, 20:1. Combination index (CI) at IC 70 was calculated by the median-effect method. Synergism and antagonism are defined as a CI mean significantly lower/higher than 1 with one-sample t -test ( P

    Journal: Nature Communications

    Article Title: Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

    doi: 10.1038/ncomms9904

    Figure Lengend Snippet: Pharmacological inhibition of Wnt/β-catenin signalling augments the cytotoxic effects of temozolomide. ( a ) Cell viability of cancer cell lines treated with increasing concentrations of different Wnt inhibitors and temozolomide (TMZ). Cell growth was assessed by FMCA or WST-1 after 72 h. All drugs were combined with a fixed molar ratio: TMZ:Celecoxib, 33:1; TMZ:G007-LK, 50:1; TMZ:LGK974, 20:1; TMZ:Wnt-C59, 20:1; TMZ:Salinomycin, 40:1; and TMZ:XAV-939, 20:1. Combination index (CI) at IC 70 was calculated by the median-effect method. Synergism and antagonism are defined as a CI mean significantly lower/higher than 1 with one-sample t -test ( P

    Article Snippet: Chemicals Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE2 (dmPGE2 ), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich).

    Techniques: Inhibition

    Genetical inhibition of Wnt/β-catenin signalling augments the cytotoxic effects of temozolomide. ( a , b ) Inhibition of β-catenin expression by shRNA or siRNA sensitizes LS174T ( a ) and SW480 ( b ) colon carcinoma cells to temozolomide. LS174T cells were grown in medium containing ±1 mg ml −1 doxycycline (doxy) and treated with increasing concentrations of temozolomide (TMZ) twice (at 0 and 48 h) for totally 96 h. Doxy-induced shRNA knockdown of β-catenin expression significantly augment the cytotoxic effect of TMZ ( t -test on log IC 50 , P =0.0003). Overexpression of MGMT reversed the cytotoxic effect of TMZ caused by β-catenin knockdown ( t -test on log IC 50 , P =0.0491). Each concentration was tested in triplicate and the experiments were repeated twice. Values are mean±s.e.m. and presented as the percentage of matched control cells. Only depletion of β-catenin inhibited cell growth to 46±5.8% (mean±s.e.m.) of untreated control. ( b ) SW480 cells were transiently transfected with siRNA against β-catenin and subsequently treated with 100 μM TMZ for 48 h. Significant growth inhibition was observed in cells treated with a combination of siRNA against β-catenin and TMZ compared with only TMZ ( t -test, P =0.002). The experiment was repeated twice. Values are mean±s.e.m. ( c ) Clonogenic capacity of LS174T cells ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ, as a single treatment or repeated treatment. The TMZ treatment was significantly more efficient in the shRNA-induced LS174T cells ( t -test, P =0.032). Each experimental point was performed in triplicate. The experiment was repeated with similar results. Mean±s.d. are displayed. ( d ) β-catenin knockdown significantly increases TMZ-induced apoptosis in LS174T cells. LS174T cells were incubated with ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ for 48 h ( t -test, P =0.036). Apoptosis was analysed by flow cytometry measurement of cells in sub-G0 phase. The experiment was repeated three times. Values are mean±s.d.

    Journal: Nature Communications

    Article Title: Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

    doi: 10.1038/ncomms9904

    Figure Lengend Snippet: Genetical inhibition of Wnt/β-catenin signalling augments the cytotoxic effects of temozolomide. ( a , b ) Inhibition of β-catenin expression by shRNA or siRNA sensitizes LS174T ( a ) and SW480 ( b ) colon carcinoma cells to temozolomide. LS174T cells were grown in medium containing ±1 mg ml −1 doxycycline (doxy) and treated with increasing concentrations of temozolomide (TMZ) twice (at 0 and 48 h) for totally 96 h. Doxy-induced shRNA knockdown of β-catenin expression significantly augment the cytotoxic effect of TMZ ( t -test on log IC 50 , P =0.0003). Overexpression of MGMT reversed the cytotoxic effect of TMZ caused by β-catenin knockdown ( t -test on log IC 50 , P =0.0491). Each concentration was tested in triplicate and the experiments were repeated twice. Values are mean±s.e.m. and presented as the percentage of matched control cells. Only depletion of β-catenin inhibited cell growth to 46±5.8% (mean±s.e.m.) of untreated control. ( b ) SW480 cells were transiently transfected with siRNA against β-catenin and subsequently treated with 100 μM TMZ for 48 h. Significant growth inhibition was observed in cells treated with a combination of siRNA against β-catenin and TMZ compared with only TMZ ( t -test, P =0.002). The experiment was repeated twice. Values are mean±s.e.m. ( c ) Clonogenic capacity of LS174T cells ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ, as a single treatment or repeated treatment. The TMZ treatment was significantly more efficient in the shRNA-induced LS174T cells ( t -test, P =0.032). Each experimental point was performed in triplicate. The experiment was repeated with similar results. Mean±s.d. are displayed. ( d ) β-catenin knockdown significantly increases TMZ-induced apoptosis in LS174T cells. LS174T cells were incubated with ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ for 48 h ( t -test, P =0.036). Apoptosis was analysed by flow cytometry measurement of cells in sub-G0 phase. The experiment was repeated three times. Values are mean±s.d.

    Article Snippet: Chemicals Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE2 (dmPGE2 ), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich).

    Techniques: Inhibition, Expressing, shRNA, Over Expression, Concentration Assay, Transfection, Incubation, Flow Cytometry, Cytometry

    Inhibition of Wnt/β-catenin in combination with temozolomide reduces tumour growth in vivo . ( a ) A combination of temozolomide and celecoxib significantly impairs the growth of established human medulloblastoma xenografts in NMRI nu/nu mice. Mice were engrafted with 7 × 10 6 D283 MED cells subcutaneously and randomized to receive either celecoxib (90 mg kg −1 ; n =12) through daily oral gastric feeding, temozolomide (7.5 mg kg −1 ; n =9; days 1–5), a combination of celecoxib and temozolomide ( n =9) or no treatment ( n =10), starting at the appearance of palpable tumours of approximately 0.10 ml (mean 0.13 ml). Celecoxib augments the inhibitory effect of temozolomide on medulloblastoma growth in vivo , as shown by the TVI (at day 12, celecoxib: TVI=6.2, P

    Journal: Nature Communications

    Article Title: Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

    doi: 10.1038/ncomms9904

    Figure Lengend Snippet: Inhibition of Wnt/β-catenin in combination with temozolomide reduces tumour growth in vivo . ( a ) A combination of temozolomide and celecoxib significantly impairs the growth of established human medulloblastoma xenografts in NMRI nu/nu mice. Mice were engrafted with 7 × 10 6 D283 MED cells subcutaneously and randomized to receive either celecoxib (90 mg kg −1 ; n =12) through daily oral gastric feeding, temozolomide (7.5 mg kg −1 ; n =9; days 1–5), a combination of celecoxib and temozolomide ( n =9) or no treatment ( n =10), starting at the appearance of palpable tumours of approximately 0.10 ml (mean 0.13 ml). Celecoxib augments the inhibitory effect of temozolomide on medulloblastoma growth in vivo , as shown by the TVI (at day 12, celecoxib: TVI=6.2, P

    Article Snippet: Chemicals Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE2 (dmPGE2 ), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich).

    Techniques: Inhibition, In Vivo, Mouse Assay

    ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.

    Journal: International Journal of Molecular Sciences

    Article Title: Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels

    doi: 10.3390/ijms19020640

    Figure Lengend Snippet: ( A ) Coomassie blue staining of the purified region-1 and region-2 of mPCSK9 proteins. Region-1 (residues 152–351) and region-2 (residues 249–452) of mouse catalytic domain were cloned and expressed by pET-3a plasmids. The proteins were reduced and purified by a reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 HPLC system. The purified proteins were analyzed by SDS/PAGE and stained with Coomassie blue. The positions of expressed proteins are shown; ( B ) MALDI mass spectrometry on Region-1. The molecular mass of purified PCSK9 region-1 in fully reduced form (PCSK9-1R). The molecular mass of PCSK9 region-1 as determined by MALDI was 21,201 Da; ( C ) MALDI mass spectrometry on Region-2. The molecular mass of purified PCSK9 region-2 in fully reduced form (PCSK9-2R). The molecular mass of PCSK9 region-2 as determined by MALDI was 21,355 Da.

    Article Snippet: The reaction was carried out at 23 °C for 24 h. Folded samples were acidified with an equal volume of 4% aqueous trifluoroacetic acid, and the non-native X-isomers were separated by HPLC using the conditions described above on an Agilent 1100 HPLC system.

    Techniques: Staining, Purification, Clone Assay, Positron Emission Tomography, High Performance Liquid Chromatography, SDS Page, Mass Spectrometry

    aps1 and pgm leaves accumulate WT ADPG content. (A) HPLC-MS/MS detection of ADPG in WT, aps1 and pgm leaves. Upper panels: Total ion chromatograms (TIC) of extracts from the indicated plants in which the selected fragmentation parent ion was 587.8 m/z. Middle panels: Extracted ion chromatograms (EIC) in which the selected ion for fragmentation of the parent ion was 346.1 m/z. Lower panels: Mass spectra (MS2) obtained from fragmentation of parent ion. ADPG was measured using an Agilent 1100 HPLC fitted with a Xbridge C18 column (100×3.0 mm I.D. particle size 3.5 µm) coupled to a MSD-Trap spectrometer (Agilent) (see Materials and Methods for further details). (B) ADPG content in WT, aps1, pgm and aps1/pgm leaves. Plants were simultaneously grown either in soil or solid MS. Leaves from 4-weeks old WT, aps1, pgm and aps1/pgm plants were simultaneously harvested after 10 h of illumination. ADPG was simultaneouly extracted from leaves of WT, aps1, pgm and aps1/pgm plants and content was simultaneously measured by HPLC-MS/MS as described in Materials and Methods . Note that, consistent with [15] , leaves of aps1, pgm and aps1/pgm plants accumulated WT ADPG content. Values represent the mean ±SD of determinations on three independent samples.

    Journal: PLoS ONE

    Article Title: HPLC-MS/MS Analyses Show That the Near-Starchless aps1 and pgm Leaves Accumulate Wild Type Levels of ADPglucose: Further Evidence for the Occurrence of Important ADPglucose Biosynthetic Pathway(s) Alternative to the pPGI-pPGM-AGP Pathway

    doi: 10.1371/journal.pone.0104997

    Figure Lengend Snippet: aps1 and pgm leaves accumulate WT ADPG content. (A) HPLC-MS/MS detection of ADPG in WT, aps1 and pgm leaves. Upper panels: Total ion chromatograms (TIC) of extracts from the indicated plants in which the selected fragmentation parent ion was 587.8 m/z. Middle panels: Extracted ion chromatograms (EIC) in which the selected ion for fragmentation of the parent ion was 346.1 m/z. Lower panels: Mass spectra (MS2) obtained from fragmentation of parent ion. ADPG was measured using an Agilent 1100 HPLC fitted with a Xbridge C18 column (100×3.0 mm I.D. particle size 3.5 µm) coupled to a MSD-Trap spectrometer (Agilent) (see Materials and Methods for further details). (B) ADPG content in WT, aps1, pgm and aps1/pgm leaves. Plants were simultaneously grown either in soil or solid MS. Leaves from 4-weeks old WT, aps1, pgm and aps1/pgm plants were simultaneously harvested after 10 h of illumination. ADPG was simultaneouly extracted from leaves of WT, aps1, pgm and aps1/pgm plants and content was simultaneously measured by HPLC-MS/MS as described in Materials and Methods . Note that, consistent with [15] , leaves of aps1, pgm and aps1/pgm plants accumulated WT ADPG content. Values represent the mean ±SD of determinations on three independent samples.

    Article Snippet: ADPG content in leaves of plants cultured on soil was measured in the Research Support Service at the Public University of Navarra using an Agilent 1100 HPLC fitted with a Xbridge C18 column (100×3.0 mm I.D. particle size 3.5 µm) coupled to a MSD-Trap spectrometer (Agilent).

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry

    The catumaxomab sample was analyzed using RP-HPLC mass spectrometry using a PLRP-S column (Varian) with an acetonitrile gradient from 27% to 42% over 40 minutes on a 1200 HPLC system (Agilent) for separation, coupled with an Agilent 6220 ESI-TOF mass

    Journal: mAbs

    Article Title: Structural and functional characterization of the trifunctional antibody catumaxomab

    doi:

    Figure Lengend Snippet: The catumaxomab sample was analyzed using RP-HPLC mass spectrometry using a PLRP-S column (Varian) with an acetonitrile gradient from 27% to 42% over 40 minutes on a 1200 HPLC system (Agilent) for separation, coupled with an Agilent 6220 ESI-TOF mass

    Article Snippet: The peptides were separated on a reversed-phase HPLC using an Agilent 1200 HPLC system equipped with a diode-array detector, an auto-sampler, and a temperature controlled column compartment (Agilent, Waldbronn, Germany).

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry

    ( a ) Representative HPLC-MS chromatogram (HILIC-ESI-MS) from site U1357, 7.15 mbsf, showing the presence of major intact polar lipid isoprenoidal glycerol dialkyl glycerol tetraethers (IPL-GDGTs) and bacterial lipids in the mass range m/z 1200–1800. The HPLC-MS chromatogram is depicted as a density map showing retention time on the x axis, m/z on the y axis and the relative peak intensity by grey shading. ( b ) Structural variety in the head groups and biphytanyl moieties of the detected IPL-GDGTs. Note that during the ether cleavage reaction the hydroxyl group in the biphytane (bp) side chain becomes dehydrated and forms biphytene (bp0:1). Position of the hydroxyl group and double bond were not determined. 1G, monohexose; 2G, dihexose; PG, phosphatidyl glycerol; HPH, hexose-phoshohexose; cren, crenarchaeol.

    Journal: The ISME Journal

    Article Title: Acetoclastic Methanosaeta are dominant methanogens in organic-rich Antarctic marine sediments

    doi: 10.1038/ismej.2017.150

    Figure Lengend Snippet: ( a ) Representative HPLC-MS chromatogram (HILIC-ESI-MS) from site U1357, 7.15 mbsf, showing the presence of major intact polar lipid isoprenoidal glycerol dialkyl glycerol tetraethers (IPL-GDGTs) and bacterial lipids in the mass range m/z 1200–1800. The HPLC-MS chromatogram is depicted as a density map showing retention time on the x axis, m/z on the y axis and the relative peak intensity by grey shading. ( b ) Structural variety in the head groups and biphytanyl moieties of the detected IPL-GDGTs. Note that during the ether cleavage reaction the hydroxyl group in the biphytane (bp) side chain becomes dehydrated and forms biphytene (bp0:1). Position of the hydroxyl group and double bond were not determined. 1G, monohexose; 2G, dihexose; PG, phosphatidyl glycerol; HPH, hexose-phoshohexose; cren, crenarchaeol.

    Article Snippet: Identification and quantification of IPLs and core GDGTs was achieved on a Agilent 1200 series HPLC system coupled to an Agilent 6520 accurate-mass quadrupole time-of-flight mass spectrometer (Summons laboratory, Agilent, Santa Clara, CA, USA) and Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Bruker maXis ultra-high-resolution orthogonal acceleration quadrupole time-of-flight tandem MS2 instrument (Hinrichs laboratory, Bruker Daltonics Inc., Billerica, MA, USA) following previously described protocols ( ; ; ; ).

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry, Hydrophilic Interaction Liquid Chromatography

    A C-type lectin was identified by TripleTOF 5600 MS. The mass spectrometer was fitted with a nanospray III ion source and coupled to an Agilent 1200 HPLC. A typical spectrum of a single peptide [(K)IFNELKAWK(D), m/z, 574.83] is shown (A). The mass and the amino acid sequence of the C-type lectin are shown and the sequence obtained from spectrometry is underlined (B). This protein was identified as Q7T228 in Uniprot database with coverage of 94%.

    Journal: PLoS ONE

    Article Title: A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

    doi: 10.1371/journal.pone.0120514

    Figure Lengend Snippet: A C-type lectin was identified by TripleTOF 5600 MS. The mass spectrometer was fitted with a nanospray III ion source and coupled to an Agilent 1200 HPLC. A typical spectrum of a single peptide [(K)IFNELKAWK(D), m/z, 574.83] is shown (A). The mass and the amino acid sequence of the C-type lectin are shown and the sequence obtained from spectrometry is underlined (B). This protein was identified as Q7T228 in Uniprot database with coverage of 94%.

    Article Snippet: Using a TripleTOF 5600 mass spectrometer coupled to an Agilent 1200 HPLC, we identified a C-type lectin from Bothrops jararacussu with 94% coverage.

    Techniques: Mass Spectrometry, High Performance Liquid Chromatography, Sequencing

    High resolution mass spectra of apo- and holo-LhaS. Spectra of apo-LhaS isolated from E. coli grown in RPMI medium (upper panel), holo-LhaS isolated from LB-medium (middle panel) and a heme standard (lower panel) were recorded on a HPLC-UV-HR mass spectrometer. The samples were diluted with MilliQ-H 2 O and applied to a Dionex Ultimate 3000 HPLC system that is coupled to the MaXis 4G ESI-QTOF mass spectrometer.

    Journal: eLife

    Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

    doi: 10.7554/eLife.57322

    Figure Lengend Snippet: High resolution mass spectra of apo- and holo-LhaS. Spectra of apo-LhaS isolated from E. coli grown in RPMI medium (upper panel), holo-LhaS isolated from LB-medium (middle panel) and a heme standard (lower panel) were recorded on a HPLC-UV-HR mass spectrometer. The samples were diluted with MilliQ-H 2 O and applied to a Dionex Ultimate 3000 HPLC system that is coupled to the MaXis 4G ESI-QTOF mass spectrometer.

    Article Snippet: The samples were diluted with MilliQ-H2 O (1:25) and 3 µL were applied to a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific), coupled to the MaXis 4G ESI-QTOF mass spectrometer (Bruker Daltonics).

    Techniques: Isolation, High Performance Liquid Chromatography, Mass Spectrometry

    T. pseudethanolicus furan aldehyde reduction. T. pseudethanolicus was grown at 65°C with 40 mM glucose and 15 mM (A) furfural or (B) 5-HMF. Growth was measured by optical density at 600 nm. Furan aldehyde concentration was measured spectrophotometrically, while furan alcohol concentration was measured by gas chromatography-mass spectrometry. Error bars are the standard deviation of three replicate cultures.

    Journal: Biotechnology for Biofuels

    Article Title: A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    doi: 10.1186/s13068-014-0165-z

    Figure Lengend Snippet: T. pseudethanolicus furan aldehyde reduction. T. pseudethanolicus was grown at 65°C with 40 mM glucose and 15 mM (A) furfural or (B) 5-HMF. Growth was measured by optical density at 600 nm. Furan aldehyde concentration was measured spectrophotometrically, while furan alcohol concentration was measured by gas chromatography-mass spectrometry. Error bars are the standard deviation of three replicate cultures.

    Article Snippet: 2,5-furandimethanol was measured using gas chromatography-mass spectrometry (GC-MS) following trimethylsilylation, with an Agilent 5975C standard quadrupole GC-MS using electron impact ionization (970 eV), as described previously [ ].

    Techniques: Concentration Assay, Gas Chromatography, Mass Spectrometry, Standard Deviation

    Chemical characterization of the barley root, maize root and chia seed exudates by gas chromatography–mass spectrometry ( GC – MS ). Error bars are the standard errors ( SEs ). Note that sugars listed are probably largely polysaccharide derived, following acid hydrolysis of the exudates.

    Journal: European Journal of Soil Science

    Article Title: Plant exudates may stabilize or weaken soil depending on species, origin and time

    doi: 10.1111/ejss.12487

    Figure Lengend Snippet: Chemical characterization of the barley root, maize root and chia seed exudates by gas chromatography–mass spectrometry ( GC – MS ). Error bars are the standard errors ( SEs ). Note that sugars listed are probably largely polysaccharide derived, following acid hydrolysis of the exudates.

    Article Snippet: Gas chromatography–mass spectrometry ( GC – MS ) analysis of exudates Analysis was carried out on an Agilent 5977B GC‐MSD fitted with an HP‐5MS, 5% phenyl, 95% dimethylpolysiloxane, 325°C column (30‐m long, 0.25‐mm internal diameter, 0.25‐μm coating) at an inlet pressure of 68.63 kPa (Agilent, Santa Clara, CA, USA).

    Techniques: Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Derivative Assay

    The Q Exactive Hybrid Quadrupole-Orbitrap-MS spectrum of compound S in positive mode.

    Journal: Molecules

    Article Title: A Simple, Rapid, and Practical Method for Distinguishing Lonicerae Japonicae Flos from Lonicerae Flos

    doi: 10.3390/molecules24193455

    Figure Lengend Snippet: The Q Exactive Hybrid Quadrupole-Orbitrap-MS spectrum of compound S in positive mode.

    Article Snippet: Identification of Compound S To identify the structure of compound S, LC-MS analysis was performed using an UltiMate 3000 UPLC system (Thermo Scientific, USA) coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, USA).

    Techniques: Mass Spectrometry

    Quantitative affinity purification mass spectrometry for comparing NvHsp70 isoform interactions under heat stress. (A) NvHsp70 isoforms were expressed with an N-terminal 6xHIS tag in yeast lacking native Hsp70s. After exposing cells to heat stress (39 °C for 2 h), chaperone interactomes were isolated by nickel-NTA magnetic bead affinity purification. Following PAGE and in-gel trypsin digestion, peptides were analyzed by Orbitrap LC–MS/MS and MassQuant informatic analysis, allowing identification of interacting proteins and determination of their relative enrichment after heat stress. Each experiment was performed in biological triplicate. (B) Expression of NvHsp70 isoforms in yeast. Lysate of cells treated as in (A) were analyzed by SDS-PAGE/Western Blotting using pan-Hsp70 and GAPDH antibodies.

    Journal: Journal of proteomics

    Article Title: Dynamic remodeling of the interactomes of Nematostella vectensis Hsp70 isoforms under heat shock

    doi: 10.1016/j.jprot.2019.103416

    Figure Lengend Snippet: Quantitative affinity purification mass spectrometry for comparing NvHsp70 isoform interactions under heat stress. (A) NvHsp70 isoforms were expressed with an N-terminal 6xHIS tag in yeast lacking native Hsp70s. After exposing cells to heat stress (39 °C for 2 h), chaperone interactomes were isolated by nickel-NTA magnetic bead affinity purification. Following PAGE and in-gel trypsin digestion, peptides were analyzed by Orbitrap LC–MS/MS and MassQuant informatic analysis, allowing identification of interacting proteins and determination of their relative enrichment after heat stress. Each experiment was performed in biological triplicate. (B) Expression of NvHsp70 isoforms in yeast. Lysate of cells treated as in (A) were analyzed by SDS-PAGE/Western Blotting using pan-Hsp70 and GAPDH antibodies.

    Article Snippet: LC-MS/MS analysis Electrospray tandem mass spectrometry (LC–MS/MS) was performed at the Mayo Clinic Proteomics Core on a Thermo Q-Exactive Orbitrap mass spectrometer, using a 70,000 RP survey scan in profile mode, m/z 340–2000 Da, with lockmasses, followed by 20 MSMS HCD fragmentation scans at 17,500 resolution on doubly and triply charged precursors.

    Techniques: Affinity Purification, Mass Spectrometry, Isolation, Polyacrylamide Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Expressing, SDS Page, Western Blot

    Trans -acylation reaction assays of various AT4 FkbB mutants after substitution of Val187 with Asp, or Cys, Ile, Lys, Trp, and Phe with allmal- and ethmal-CoA as substrates and the Production of FK506 and FK520 in WT, YN06-01, and YN06-02. (A) The change of transferring allmal/ethmal unit to ACP10 FkbA in trans -acylation reactions by AT4 FkbB and its mutants V187D, V187C, V187I, V187K, V187W, and V187F. HPLC analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (B) and in the absence of AT4 FkbB (D) . MS analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (C) and in the absence of AT4 FkbB (E) . The peaks were assigned as follows: b 0 , holo-ACP10 FkbA ; b 1 , allmal-ACP10 FkbA ; b 2 , ethmal-ACP10 FkbA . (F) Production of FK506 and FK520 in WT, YN06-01 and YN06-02. Analyses of variance were conducted to determine the difference of WT and mutants using SPSS 20. The LSD multiple range tests were evaluated for significant differences among WT and mutants ( P

    Journal: Frontiers in Microbiology

    Article Title: Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups

    doi: 10.3389/fmicb.2018.01840

    Figure Lengend Snippet: Trans -acylation reaction assays of various AT4 FkbB mutants after substitution of Val187 with Asp, or Cys, Ile, Lys, Trp, and Phe with allmal- and ethmal-CoA as substrates and the Production of FK506 and FK520 in WT, YN06-01, and YN06-02. (A) The change of transferring allmal/ethmal unit to ACP10 FkbA in trans -acylation reactions by AT4 FkbB and its mutants V187D, V187C, V187I, V187K, V187W, and V187F. HPLC analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (B) and in the absence of AT4 FkbB (D) . MS analyses of transferring allmal-CoA : ethmal-CoA = 1:1 to holo-ACP10 FkbA in the presence of V187K (C) and in the absence of AT4 FkbB (E) . The peaks were assigned as follows: b 0 , holo-ACP10 FkbA ; b 1 , allmal-ACP10 FkbA ; b 2 , ethmal-ACP10 FkbA . (F) Production of FK506 and FK520 in WT, YN06-01 and YN06-02. Analyses of variance were conducted to determine the difference of WT and mutants using SPSS 20. The LSD multiple range tests were evaluated for significant differences among WT and mutants ( P

    Article Snippet: The concentration of FK506 and FK520 was determined using an HPLC system (Agilent Series 1100, Agilent) equipped with a SB-C18 column (150 mm × 2.1 mm, Agilent).

    Techniques: Transferring, High Performance Liquid Chromatography, Mass Spectrometry

    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay, Software, Two Tailed Test, Produced, Concentration Assay

    Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay, Pyrophosphate Assay

    Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay

    Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Incubation, Software, Injection, Fluorescence In Situ Hybridization, Inhibition, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Representative standard curves of the Hepcidin-25H calibration. (A) A linear response of Hepcidin-25H quantitation between 0.5 to 6 picomoles was demonstrated in TSQ Quantum Ultra, (B) The linear response of the Hepcidin-25H quantification between 100 to 500 femtomoles was demonstrated in LTQ-XL linear ion trap instrument.

    Journal: Clinica chimica acta; international journal of clinical chemistry

    Article Title: Effects of a Single Dose of Oral Iron on Hepcidin Concentrations in Human Urine and Serum Analyzed by a Robust LC-MS/MS Method

    doi: 10.1016/j.cca.2011.08.014

    Figure Lengend Snippet: Representative standard curves of the Hepcidin-25H calibration. (A) A linear response of Hepcidin-25H quantitation between 0.5 to 6 picomoles was demonstrated in TSQ Quantum Ultra, (B) The linear response of the Hepcidin-25H quantification between 100 to 500 femtomoles was demonstrated in LTQ-XL linear ion trap instrument.

    Article Snippet: LC-MS/MS was performed on custom nanoflow-split Waters ACQUITY Ultra-Performance Liquid Chromatography (UPLC) coupled to a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer and/or a Thermo LTQ linear ion trap mass spectrometer equipped with a New-Objective electrospray ionization source.

    Techniques: Quantitation Assay

    Metabolic Labeling and Workflow. ( A ) Glucose, acetate, fatty acids, and amino acids produce acetyl-CoA for use in acetylating cytoplasmic and nuclear proteins. The thicker arrows indicate that glucose contributes more to the production of acetyl-coA that subsequently acetylates proteins, compared to acetate. ( B ) The workflow consisted of growing HeLa cells in heavy-labeled media, collecting samples at eight time points, lysing the cells, digesting the proteins, enriching for acetylated peptides, and analyzing the peptides by mass spectrometry. The Orbitrap image is adapted from Thermo Fisher Scientific 56 . The cartoon cell matter and lab equipment were slightly modified from Servier Medical Art 57 .

    Journal: Scientific Reports

    Article Title: Proteome-wide acetylation dynamics in human cells

    doi: 10.1038/s41598-017-09918-3

    Figure Lengend Snippet: Metabolic Labeling and Workflow. ( A ) Glucose, acetate, fatty acids, and amino acids produce acetyl-CoA for use in acetylating cytoplasmic and nuclear proteins. The thicker arrows indicate that glucose contributes more to the production of acetyl-coA that subsequently acetylates proteins, compared to acetate. ( B ) The workflow consisted of growing HeLa cells in heavy-labeled media, collecting samples at eight time points, lysing the cells, digesting the proteins, enriching for acetylated peptides, and analyzing the peptides by mass spectrometry. The Orbitrap image is adapted from Thermo Fisher Scientific 56 . The cartoon cell matter and lab equipment were slightly modified from Servier Medical Art 57 .

    Article Snippet: Acetylated peptides were then enriched using anti-acetyl-lysine antibodies and run on nano liquid chromatography coupled online with tandem mass spectrometry (nanoLC-MS/MS) on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), which allows for detection of isotope incorporation into acetylated proteins.

    Techniques: Labeling, Mass Spectrometry, Modification

    HPLC-electrospray (ESI)-negative mass spectrum of metabolites of SKN dependent on NADPH, UDPGA, or both cofactors. Mass spectrum of SKN ( A ), mass spectrum of M1 ( B ), mass spectrum of M2 ( C ), mass spectrum of M3 ( D ), mass spectrum of M4 ( E ), mass spectrum of M5 ( F ), and mass spectrum of M6 ( G ) are shown. The metabolites are identified in Table 3 .

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases

    doi: 10.3390/molecules23071542

    Figure Lengend Snippet: HPLC-electrospray (ESI)-negative mass spectrum of metabolites of SKN dependent on NADPH, UDPGA, or both cofactors. Mass spectrum of SKN ( A ), mass spectrum of M1 ( B ), mass spectrum of M2 ( C ), mass spectrum of M3 ( D ), mass spectrum of M4 ( E ), mass spectrum of M5 ( F ), and mass spectrum of M6 ( G ) are shown. The metabolites are identified in Table 3 .

    Article Snippet: LC-ESI-MS was performed with an LTQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA, USA) with an ESI source, which was coupled with the Agilent HPLC system.

    Techniques: High Performance Liquid Chromatography

    Estimated power versus putative type 1 error rate (α) with validation (Progenesis PPA measurements). a , power results for Progenesis PPA measurement data (as in ). At α = 0.05, the estimated power of ProPCA is 0.60, the estimated

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Increased Power for the Analysis of Label-free LC-MS/MS Proteomics Data by Combining Spectral Counts and Peptide Peak Attributes *

    doi: 10.1074/mcp.M110.002774

    Figure Lengend Snippet: Estimated power versus putative type 1 error rate (α) with validation (Progenesis PPA measurements). a , power results for Progenesis PPA measurement data (as in ). At α = 0.05, the estimated power of ProPCA is 0.60, the estimated

    Article Snippet: The msInspect/AMT peak alignment algorithm has been described ( , , ); the Progenesis LC-MS software utilizes a proprietary alignment algorithm.

    Techniques: