liquid chromatography tandem mass spectrometry  (Thermo Fisher)


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    Structured Review

    Thermo Fisher liquid chromatography tandem mass spectrometry
    LY3031207 and proposed structures for metabolites in human plasma and urine (dotted line indicates formation may involve multiple steps). Plasma and urine samples were prepared as Hamilton AUC 0–24h . Metabolites were identified using accurate <t>mass</t> <t>liquid</t> <t>chromatography/mass</t> <t>spectrometry</t> and liquid chromatography/mass spectrometry/mass spectometry with positive electrospray as well as metabolite retention time comparisons. Concentration estimates for each metabolite in the extracted ion chromatograms were reported as relative to the mass spectrometric peak height of LY3031207. M1–6, metabolites M1–6
    Liquid Chromatography Tandem Mass Spectrometry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    liquid chromatography tandem mass spectrometry - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor) Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor"

    Article Title: Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor) Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor

    Journal: British Journal of Clinical Pharmacology

    doi: 10.1111/bcp.13423

    LY3031207 and proposed structures for metabolites in human plasma and urine (dotted line indicates formation may involve multiple steps). Plasma and urine samples were prepared as Hamilton AUC 0–24h . Metabolites were identified using accurate mass liquid chromatography/mass spectrometry and liquid chromatography/mass spectrometry/mass spectometry with positive electrospray as well as metabolite retention time comparisons. Concentration estimates for each metabolite in the extracted ion chromatograms were reported as relative to the mass spectrometric peak height of LY3031207. M1–6, metabolites M1–6
    Figure Legend Snippet: LY3031207 and proposed structures for metabolites in human plasma and urine (dotted line indicates formation may involve multiple steps). Plasma and urine samples were prepared as Hamilton AUC 0–24h . Metabolites were identified using accurate mass liquid chromatography/mass spectrometry and liquid chromatography/mass spectrometry/mass spectometry with positive electrospray as well as metabolite retention time comparisons. Concentration estimates for each metabolite in the extracted ion chromatograms were reported as relative to the mass spectrometric peak height of LY3031207. M1–6, metabolites M1–6

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Concentration Assay

    2) Product Images from "Pharmacogenetics Meets Metabolomics: Discovery of Tryptophan as a New Endogenous OCT2 Substrate Related to Metformin Disposition"

    Article Title: Pharmacogenetics Meets Metabolomics: Discovery of Tryptophan as a New Endogenous OCT2 Substrate Related to Metformin Disposition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036637

    Absolute levels of tryptophan in urine samples. Tryptophan levels were measured from SLC22A2 reference ( n = 9), 808GT ( n = 6), and 808TT ( n = 6) carriers using selected reaction monitoring with liquid chromatography-coupled tandem mass spectrometry. The boundary of the box closest to zero indicates the 25 th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75 th percentile.
    Figure Legend Snippet: Absolute levels of tryptophan in urine samples. Tryptophan levels were measured from SLC22A2 reference ( n = 9), 808GT ( n = 6), and 808TT ( n = 6) carriers using selected reaction monitoring with liquid chromatography-coupled tandem mass spectrometry. The boundary of the box closest to zero indicates the 25 th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75 th percentile.

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    3) Product Images from "Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays"

    Article Title: Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143639

    Liquid chromatography–tandem mass spectrometry chromatogram in the multiple-reaction monitoring mode for the dried blood spots.
    Figure Legend Snippet: Liquid chromatography–tandem mass spectrometry chromatogram in the multiple-reaction monitoring mode for the dried blood spots.

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    4) Product Images from "Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid"

    Article Title: Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid

    Journal: Frontiers in Nutrition

    doi: 10.3389/fnut.2017.00009

    Liquid chromatography–tandem mass spectrometry chromatogram of calibrator 14 (Table 2 ) .
    Figure Legend Snippet: Liquid chromatography–tandem mass spectrometry chromatogram of calibrator 14 (Table 2 ) .

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    Liquid chromatography–tandem mass spectrometry chromatogram of the enzyme mixture (75 μL rat serum and 500 μL chicken pancreas suspension) .
    Figure Legend Snippet: Liquid chromatography–tandem mass spectrometry chromatogram of the enzyme mixture (75 μL rat serum and 500 μL chicken pancreas suspension) .

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    Liquid chromatography–tandem mass spectrometry chromatogram in the multiple-reaction monitoring mode for volumetric absorptive microsamplers [10.8 μL whole blood sample (329 nmol L −1 5-CH 3 -H 4 folate)] .
    Figure Legend Snippet: Liquid chromatography–tandem mass spectrometry chromatogram in the multiple-reaction monitoring mode for volumetric absorptive microsamplers [10.8 μL whole blood sample (329 nmol L −1 5-CH 3 -H 4 folate)] .

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    Liquid chromatography–tandem mass spectrometry chromatogram of a water blank .
    Figure Legend Snippet: Liquid chromatography–tandem mass spectrometry chromatogram of a water blank .

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    5) Product Images from "Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib"

    Article Title: Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib

    Journal: Journal of Proteomics

    doi: 10.1016/j.jprot.2017.08.015

    Experimental outline and phosphoprotome dataset overview. (A) Dose response curve of A204 parental and PazR cells to pazopanib and A204 parental and DasR cells to dasatinib respectively. (B) Schematic of sample preparation workflow. Pazopanib and dasatinib resistant A204 cell lines (PazR DasR respectively) were generated and heavy SILAC labelled as previously described [12] . A204 parental cells were light SILAC labelled. After cell lysis, either heavy PazR or DasR were mixed 1:1 with light A204 parental lysate then reduced, alkylated and trypsin digested. The resulting peptides underwent phospho-tyrosine (pTyr) peptide immunoprecipitation, data previously published [12] . The supernatant from the immunoprecipitation was further enriched with immobilised metal affinity chromatography (IMAC) or titanium dioxide (TiO 2 ) prior to liquid chromatography tandem mass spectrometry analysis (LC MS/MS). (C) Venn diagrams show distribution of phosphorylation sites across three biological replicates (R1, R2 and R3) in PazR/A204 and DasR/A204 experiments.
    Figure Legend Snippet: Experimental outline and phosphoprotome dataset overview. (A) Dose response curve of A204 parental and PazR cells to pazopanib and A204 parental and DasR cells to dasatinib respectively. (B) Schematic of sample preparation workflow. Pazopanib and dasatinib resistant A204 cell lines (PazR DasR respectively) were generated and heavy SILAC labelled as previously described [12] . A204 parental cells were light SILAC labelled. After cell lysis, either heavy PazR or DasR were mixed 1:1 with light A204 parental lysate then reduced, alkylated and trypsin digested. The resulting peptides underwent phospho-tyrosine (pTyr) peptide immunoprecipitation, data previously published [12] . The supernatant from the immunoprecipitation was further enriched with immobilised metal affinity chromatography (IMAC) or titanium dioxide (TiO 2 ) prior to liquid chromatography tandem mass spectrometry analysis (LC MS/MS). (C) Venn diagrams show distribution of phosphorylation sites across three biological replicates (R1, R2 and R3) in PazR/A204 and DasR/A204 experiments.

    Techniques Used: Sample Prep, Generated, Lysis, Immunoprecipitation, Affinity Chromatography, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    6) Product Images from "Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma"

    Article Title: Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0238251

    The flowchart of the study design. CCA, cholangiocarcinoma; IgG, immunoglobulin G; HCC, hepatocellular carcinoma; LC-MS/MS, liquid chromatography–tandem mass spectrometry.
    Figure Legend Snippet: The flowchart of the study design. CCA, cholangiocarcinoma; IgG, immunoglobulin G; HCC, hepatocellular carcinoma; LC-MS/MS, liquid chromatography–tandem mass spectrometry.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry

    7) Product Images from "Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite"

    Article Title: Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-015-0381-7

    MEP pathway flux is not affected by RpoS. a Intracellular MEP pathway metabolite pools during lycopene production in MG1655* and Seq + with dxs overexpression. Glyceraldehyde-3phosphate (GA3P), dihydroxyacetone-phosphate (DHAP), pyruvate (PYR), 1-deoxy- d -xylulose 5-phosphate (DXP), 2-C-methyl- d -erythritol-4-phosphate (MEP), 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME), 2-C-methyl- d -erythritol-2,4-cyclodiphosphate (MEcPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP; no chromatographic separation of IPP and DMAPP) and farnesyl pyrophosphate (FPP) were quantified using liquid chromatography tandem mass spectrometry (LC–MS/MS). 4-Diphosphocytidyl-2-C-methyl- d -erythritol-2-phosphate (CDP-MEP) was below detection level in all samples. b Isoprene production in the two strains harboring pT-ispS(L70R)
    Figure Legend Snippet: MEP pathway flux is not affected by RpoS. a Intracellular MEP pathway metabolite pools during lycopene production in MG1655* and Seq + with dxs overexpression. Glyceraldehyde-3phosphate (GA3P), dihydroxyacetone-phosphate (DHAP), pyruvate (PYR), 1-deoxy- d -xylulose 5-phosphate (DXP), 2-C-methyl- d -erythritol-4-phosphate (MEP), 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME), 2-C-methyl- d -erythritol-2,4-cyclodiphosphate (MEcPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP; no chromatographic separation of IPP and DMAPP) and farnesyl pyrophosphate (FPP) were quantified using liquid chromatography tandem mass spectrometry (LC–MS/MS). 4-Diphosphocytidyl-2-C-methyl- d -erythritol-2-phosphate (CDP-MEP) was below detection level in all samples. b Isoprene production in the two strains harboring pT-ispS(L70R)

    Techniques Used: Over Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    8) Product Images from "Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components"

    Article Title: Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components

    Journal: Proteome Science

    doi: 10.1186/1477-5956-9-39

    Experimental design and workflow for the quantitative analysis of Spirulina in response to immediate temperature reduction . (A) Experimental design to study the response of Spirulina to low-temperature stress represented by differentially expressed proteins in three subcellular fractions. (B) An illustration of the quantitative proteomic iTRAQ (4-plex)-multidimensional liquid chromatography-tandem mass spectrometry workflow.
    Figure Legend Snippet: Experimental design and workflow for the quantitative analysis of Spirulina in response to immediate temperature reduction . (A) Experimental design to study the response of Spirulina to low-temperature stress represented by differentially expressed proteins in three subcellular fractions. (B) An illustration of the quantitative proteomic iTRAQ (4-plex)-multidimensional liquid chromatography-tandem mass spectrometry workflow.

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    9) Product Images from "Quorum Sensing Controls Biofilm Formation in Vibrio cholerae through Modulation of Cyclic Di-GMP Levels and Repression of vpsT "

    Article Title: Quorum Sensing Controls Biofilm Formation in Vibrio cholerae through Modulation of Cyclic Di-GMP Levels and Repression of vpsT

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01756-07

    c-di-GMP levels in V. cholerae strains. (A) In vivo c-di-GMP levels were determined by measuring ionization fragments with SRM1 ( m/z 689→344 at 32 eV [left]) and SRM2 ( m/z 689→150 at 45 eV [right]). The top panels show the purified standard at 2 μg/ml in 50:50 methanol-H 2 O, and the bottom panels show the biological sample. c-di-GMP has a retention time of 27.5 min. The peak labeled with an asterisk in the biological sample is an unknown interfering compound that is neither a conformer nor an isomer of c-di-GMP. (B) Relative levels of c-di-GMP in V. cholerae WT ( ) and the luxO ( D47E ) mutant (▪) containing a vector control or overexpressing QrgB, QrgB* (GGEEF→AAEEF), VC1086, or VC1086* (EAL→AAL) were determined by tandem high-pressure liquid chromatography-mass spectrophotometric analysis. Each strain was analyzed in triplicate. Error bars indicate the standard errors of the mean.
    Figure Legend Snippet: c-di-GMP levels in V. cholerae strains. (A) In vivo c-di-GMP levels were determined by measuring ionization fragments with SRM1 ( m/z 689→344 at 32 eV [left]) and SRM2 ( m/z 689→150 at 45 eV [right]). The top panels show the purified standard at 2 μg/ml in 50:50 methanol-H 2 O, and the bottom panels show the biological sample. c-di-GMP has a retention time of 27.5 min. The peak labeled with an asterisk in the biological sample is an unknown interfering compound that is neither a conformer nor an isomer of c-di-GMP. (B) Relative levels of c-di-GMP in V. cholerae WT ( ) and the luxO ( D47E ) mutant (▪) containing a vector control or overexpressing QrgB, QrgB* (GGEEF→AAEEF), VC1086, or VC1086* (EAL→AAL) were determined by tandem high-pressure liquid chromatography-mass spectrophotometric analysis. Each strain was analyzed in triplicate. Error bars indicate the standard errors of the mean.

    Techniques Used: In Vivo, Purification, Labeling, Mutagenesis, Plasmid Preparation, High Performance Liquid Chromatography

    10) Product Images from "Isocitrate dehydrogenase mutations confer dasatinib hypersensitivity and SRC-dependence in intrahepatic cholangiocarcinoma"

    Article Title: Isocitrate dehydrogenase mutations confer dasatinib hypersensitivity and SRC-dependence in intrahepatic cholangiocarcinoma

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-15-1442

    Identification of dasatinib targets in IDHm ICC A. Immunoblot demonstrating that dasatinib causes loss of phospho-p70 S6 Kinase (Thr389) and phospho-S6 (Ser235/236) specifically in IDHm ICC cells. Neither IDHm nor IDH WT ICC show dasatinib-induced changes in phospho-ERK1/2 (Thr202/Tyr204), phosph-STAT3 (Ser727), BCL2, or MCL1. B. Schematic illustrating methods used to characterize dynamic changes in the “active kinome” of ICC cells treated with dasatinib. IDH WT or IDHm ICC lines were treated with dasatinib (20 nM) or vehicle control for one hour prior to harvest. Whole cell lysates were run over a multiplexed kinase inhibitor bead (MIB) column containing a panel of sepharose beads covalently linked to 12 kinase inhibitors with distinct specificity profiles. Due to their preferential binding to active kinases, inactive kinases are discarded in the flow-through while active kinases were isolated in the eluent. The eluent was then subjected to tryptic digestion and peptide identification through liquid chromatography-tandem mass spectrometry (LC-MS/MS). To identify active kinases potently inhibited by dasatinib in each cell line, the active kinome of dasatinib-treated cells was compared to that of vehicle control. C. Heat map of kinases enriched in the active kinome of dasatinib-treated cells compared to vehicle control cells (log 2 ratio is shown). Kinases that are active at baseline, inhibited > 75% by dasatinib, and are common to both IDHm ICC are in blue.
    Figure Legend Snippet: Identification of dasatinib targets in IDHm ICC A. Immunoblot demonstrating that dasatinib causes loss of phospho-p70 S6 Kinase (Thr389) and phospho-S6 (Ser235/236) specifically in IDHm ICC cells. Neither IDHm nor IDH WT ICC show dasatinib-induced changes in phospho-ERK1/2 (Thr202/Tyr204), phosph-STAT3 (Ser727), BCL2, or MCL1. B. Schematic illustrating methods used to characterize dynamic changes in the “active kinome” of ICC cells treated with dasatinib. IDH WT or IDHm ICC lines were treated with dasatinib (20 nM) or vehicle control for one hour prior to harvest. Whole cell lysates were run over a multiplexed kinase inhibitor bead (MIB) column containing a panel of sepharose beads covalently linked to 12 kinase inhibitors with distinct specificity profiles. Due to their preferential binding to active kinases, inactive kinases are discarded in the flow-through while active kinases were isolated in the eluent. The eluent was then subjected to tryptic digestion and peptide identification through liquid chromatography-tandem mass spectrometry (LC-MS/MS). To identify active kinases potently inhibited by dasatinib in each cell line, the active kinome of dasatinib-treated cells was compared to that of vehicle control. C. Heat map of kinases enriched in the active kinome of dasatinib-treated cells compared to vehicle control cells (log 2 ratio is shown). Kinases that are active at baseline, inhibited > 75% by dasatinib, and are common to both IDHm ICC are in blue.

    Techniques Used: Immunocytochemistry, Binding Assay, Flow Cytometry, Isolation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    11) Product Images from "Homocysteinethiolactone and Paraoxonase"

    Article Title: Homocysteinethiolactone and Paraoxonase

    Journal: Diabetes Care

    doi: 10.2337/dc10-0132

    PON activity and HCTL levels in the vitreous of PDR and MH case subjects. Distribution graphs show the reciprocal relationship of HCTLase and AREase in PDR ( n = 13) and MH ( n = 8). A : PON-AREase activity. B : PON-HCTLase activity. C : Activity staining for PON protein in the vitreous using phenylacetate as substrate and parasoaniline as chromogen. The band was observed at 66 kDa ( lane 1: MH; lane s 2–4 : PDR; lane 5 : high–molecular weight marker). Representative liquid chromatography–tandem mass spectrometry chromatogram showing the HCTL ( left ) and the corresponding internal standard, namely homatropine ( right ). D : Standard vitreous. E : MH vitreous. F : PDR vitreous. The m/z of HCTL is 118.2 and homatroprine is 276.1 (seen as the peak). G : Distribution of HCTL levels in PDR ( n = 9) and MH ( n = 3) case subjects. Correlation between HCTL and PON-HCTLase is shown. H : PDR ( n = 9), ♦; MH ( n = 3), ●. (A high-quality color representation of this figure is available in the online issue.)
    Figure Legend Snippet: PON activity and HCTL levels in the vitreous of PDR and MH case subjects. Distribution graphs show the reciprocal relationship of HCTLase and AREase in PDR ( n = 13) and MH ( n = 8). A : PON-AREase activity. B : PON-HCTLase activity. C : Activity staining for PON protein in the vitreous using phenylacetate as substrate and parasoaniline as chromogen. The band was observed at 66 kDa ( lane 1: MH; lane s 2–4 : PDR; lane 5 : high–molecular weight marker). Representative liquid chromatography–tandem mass spectrometry chromatogram showing the HCTL ( left ) and the corresponding internal standard, namely homatropine ( right ). D : Standard vitreous. E : MH vitreous. F : PDR vitreous. The m/z of HCTL is 118.2 and homatroprine is 276.1 (seen as the peak). G : Distribution of HCTL levels in PDR ( n = 9) and MH ( n = 3) case subjects. Correlation between HCTL and PON-HCTLase is shown. H : PDR ( n = 9), ♦; MH ( n = 3), ●. (A high-quality color representation of this figure is available in the online issue.)

    Techniques Used: Activity Assay, Staining, Molecular Weight, Marker, Liquid Chromatography, Mass Spectrometry

    12) Product Images from "Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart"

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111448

    Mitochondrial GAPDH and total GAPDH levels following cardioprotection. ( A ) GAPDH protein levels in the mitochondrial fraction of hearts subjected to control or an IPC-I/R protocol were assessed via liquid chromatography-tandem mass spectrometry, followed by label-free peptide quantification (*p
    Figure Legend Snippet: Mitochondrial GAPDH and total GAPDH levels following cardioprotection. ( A ) GAPDH protein levels in the mitochondrial fraction of hearts subjected to control or an IPC-I/R protocol were assessed via liquid chromatography-tandem mass spectrometry, followed by label-free peptide quantification (*p

    Techniques Used: Liquid Chromatography, Mass Spectrometry

    13) Product Images from "Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc"

    Article Title: Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc

    Journal: Asian Spine Journal

    doi: 10.31616/asj.2019.0217

    General workflow of protein extraction and identification. S1, soluble fraction; S2, insoluble fraction; SDR-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; LC, liquid chromatography; MS/MS, tandem mass spectrometry; PD, proteome discoverer.
    Figure Legend Snippet: General workflow of protein extraction and identification. S1, soluble fraction; S2, insoluble fraction; SDR-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; LC, liquid chromatography; MS/MS, tandem mass spectrometry; PD, proteome discoverer.

    Techniques Used: Protein Extraction, Polyacrylamide Gel Electrophoresis, Liquid Chromatography, Tandem Mass Spectroscopy, Mass Spectrometry

    14) Product Images from "Di-Adenosine Tetraphosphate (Ap4A) Metabolism Impacts Biofilm Formation by Pseudomonas fluorescens via Modulation of c-di-GMP-Dependent Pathways ▿"

    Article Title: Di-Adenosine Tetraphosphate (Ap4A) Metabolism Impacts Biofilm Formation by Pseudomonas fluorescens via Modulation of c-di-GMP-Dependent Pathways ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01571-09

    Effect of the apaH mutation on nucleotide pools. (A) Analysis of nucleotide pools by mass spectrometry. Wild-type (Wt) and apaH mutant cultures were grown in triplicate for 5 to 6 h in high-P i (K10T-1) medium before extraction of nucleotides and relative quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extracts were analyzed to determine the levels of ADP, ATP, CTP, UTP, GTP, c-di-GMP, and Ap4A. The error bars indicate standard errors ( n = 3). For statistical inference with Student's t tests, alpha (α) was set at 0.0071 after adjustment for multiple comparisons using a Bonferroni correction (α = 0.05/7). Statistically significant differences are indicated by an asterisk. Ap4A could not be detected in wild-type extracts. Using this assay, the limit of detection for Ap4A is ∼20 ng/ml. (B) Assay for c-di-GMP phosphodiesterase activity. Purified ApaH (5 mg/ml) was unable to cleave c-di-GMP. SadR was used as a positive control for PDE activity, which completely cleaved c-di-GMP to generate pGpG.
    Figure Legend Snippet: Effect of the apaH mutation on nucleotide pools. (A) Analysis of nucleotide pools by mass spectrometry. Wild-type (Wt) and apaH mutant cultures were grown in triplicate for 5 to 6 h in high-P i (K10T-1) medium before extraction of nucleotides and relative quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extracts were analyzed to determine the levels of ADP, ATP, CTP, UTP, GTP, c-di-GMP, and Ap4A. The error bars indicate standard errors ( n = 3). For statistical inference with Student's t tests, alpha (α) was set at 0.0071 after adjustment for multiple comparisons using a Bonferroni correction (α = 0.05/7). Statistically significant differences are indicated by an asterisk. Ap4A could not be detected in wild-type extracts. Using this assay, the limit of detection for Ap4A is ∼20 ng/ml. (B) Assay for c-di-GMP phosphodiesterase activity. Purified ApaH (5 mg/ml) was unable to cleave c-di-GMP. SadR was used as a positive control for PDE activity, which completely cleaved c-di-GMP to generate pGpG.

    Techniques Used: Mutagenesis, Mass Spectrometry, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Purification, Positive Control

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry Samples were measured by means of LC–MS/MS (Finnigan Surveyor Plus HPLC System and Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer; Thermo Electron Corporation, Waltham, MA, USA). .. Analytes were separated on a Nucleosil 100–5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany).

    Article Title: Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry About 10 μL of the samples were measured using a Finnigan Surveyor Plus HPLC System coupled with a Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer (Thermo Electron Corporation, Waltham, MA, USA) equipped with a Nucleosil 100-5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany). .. Aqueous formic acid (0.1%; eluent A) and acetonitrile containing 0.1% formic acid (eluent B) were used as eluents at a flow rate of 0.3 mL/min.

    Sample Prep:

    Article Title: Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma
    Article Snippet: .. Sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis Five microliters of plasma were diluted with phosphate-buffered saline and incubated with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific, Waltham, MA, USA) and Protein G-sepharose beads (GE Healthcare, Piscataway, NJ, USA) to remove albumin and immunoglobulin (Ig) G. Proteins not bound to the beads were harvested, denatured using 10% sodium dodecyl sulfate plus 10 mM dithiothreitol at 95°C for 10 minutes, and alkylated with 10 mM iodoacetamide at 37°C in dark for 1 hour. .. The proteins were then cleaned and concentrated using Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff: 3,000 Da) device (Merck Millipore, Darmstadt, Germany) and digested at 37°C overnight using sequencing grade trypsin (Promega, Fitchburg, WI, USA) in 10 mM ammonium bicarbonate in an enzyme-to-substrate ratio of 1:50.

    Chromatography:

    Article Title: Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components
    Article Snippet: .. After the twenty-four off-gel fractions were cleaned using ZipTips® (Millipore, USA), the samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Famos-Switchos-Ultimate nano-LC system (Dionex, LC packing, The Netherlands) interfaced with a QSTAR XL (Applied Biosystems; MDS-Sciex) tandem ESI-QUAD-TOF MS. .. Concentrated off-gel fractions were resuspended in 0.1% formic acid, injected and captured in a 0.3 × 5 mm C18 trapping cartridge (LC Packings).

    Article Title: Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma
    Article Snippet: .. Sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis Five microliters of plasma were diluted with phosphate-buffered saline and incubated with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific, Waltham, MA, USA) and Protein G-sepharose beads (GE Healthcare, Piscataway, NJ, USA) to remove albumin and immunoglobulin (Ig) G. Proteins not bound to the beads were harvested, denatured using 10% sodium dodecyl sulfate plus 10 mM dithiothreitol at 95°C for 10 minutes, and alkylated with 10 mM iodoacetamide at 37°C in dark for 1 hour. .. The proteins were then cleaned and concentrated using Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff: 3,000 Da) device (Merck Millipore, Darmstadt, Germany) and digested at 37°C overnight using sequencing grade trypsin (Promega, Fitchburg, WI, USA) in 10 mM ammonium bicarbonate in an enzyme-to-substrate ratio of 1:50.

    Article Title: Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry Samples were measured by means of LC–MS/MS (Finnigan Surveyor Plus HPLC System and Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer; Thermo Electron Corporation, Waltham, MA, USA). .. Analytes were separated on a Nucleosil 100–5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany).

    Article Title: Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry About 10 μL of the samples were measured using a Finnigan Surveyor Plus HPLC System coupled with a Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer (Thermo Electron Corporation, Waltham, MA, USA) equipped with a Nucleosil 100-5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany). .. Aqueous formic acid (0.1%; eluent A) and acetonitrile containing 0.1% formic acid (eluent B) were used as eluents at a flow rate of 0.3 mL/min.

    Article Title: Pharmacogenetics Meets Metabolomics: Discovery of Tryptophan as a New Endogenous OCT2 Substrate Related to Metformin Disposition
    Article Snippet: .. Assay of tryptophan concentrations using liquid chromatography-tandem mass spectrometry Tryptophan concentrations in urine samples were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS; API 3000; Applied Biosystems, Foster City, CA). ..

    Article Title: Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor) Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor
    Article Snippet: .. Putative metabolite structures were identified using accurate mass liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry with positive electrospray ionization (LTQ Orbitrap XL, Thermo Scientific, Waltham, MA, USA) as well as metabolite retention time comparisons. ..

    Article Title: Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib
    Article Snippet: .. 2.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) For IMAC-enriched samples, reversed phase chromatography was performed on eluted peptides using a Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific). ..

    Incubation:

    Article Title: Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma
    Article Snippet: .. Sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis Five microliters of plasma were diluted with phosphate-buffered saline and incubated with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific, Waltham, MA, USA) and Protein G-sepharose beads (GE Healthcare, Piscataway, NJ, USA) to remove albumin and immunoglobulin (Ig) G. Proteins not bound to the beads were harvested, denatured using 10% sodium dodecyl sulfate plus 10 mM dithiothreitol at 95°C for 10 minutes, and alkylated with 10 mM iodoacetamide at 37°C in dark for 1 hour. .. The proteins were then cleaned and concentrated using Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff: 3,000 Da) device (Merck Millipore, Darmstadt, Germany) and digested at 37°C overnight using sequencing grade trypsin (Promega, Fitchburg, WI, USA) in 10 mM ammonium bicarbonate in an enzyme-to-substrate ratio of 1:50.

    Mass Spectrometry:

    Article Title: Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components
    Article Snippet: .. After the twenty-four off-gel fractions were cleaned using ZipTips® (Millipore, USA), the samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Famos-Switchos-Ultimate nano-LC system (Dionex, LC packing, The Netherlands) interfaced with a QSTAR XL (Applied Biosystems; MDS-Sciex) tandem ESI-QUAD-TOF MS. .. Concentrated off-gel fractions were resuspended in 0.1% formic acid, injected and captured in a 0.3 × 5 mm C18 trapping cartridge (LC Packings).

    Article Title: Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma
    Article Snippet: .. Sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis Five microliters of plasma were diluted with phosphate-buffered saline and incubated with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific, Waltham, MA, USA) and Protein G-sepharose beads (GE Healthcare, Piscataway, NJ, USA) to remove albumin and immunoglobulin (Ig) G. Proteins not bound to the beads were harvested, denatured using 10% sodium dodecyl sulfate plus 10 mM dithiothreitol at 95°C for 10 minutes, and alkylated with 10 mM iodoacetamide at 37°C in dark for 1 hour. .. The proteins were then cleaned and concentrated using Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff: 3,000 Da) device (Merck Millipore, Darmstadt, Germany) and digested at 37°C overnight using sequencing grade trypsin (Promega, Fitchburg, WI, USA) in 10 mM ammonium bicarbonate in an enzyme-to-substrate ratio of 1:50.

    Article Title: Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry Samples were measured by means of LC–MS/MS (Finnigan Surveyor Plus HPLC System and Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer; Thermo Electron Corporation, Waltham, MA, USA). .. Analytes were separated on a Nucleosil 100–5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany).

    Article Title: Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid
    Article Snippet: .. Liquid Chromatography–Tandem Mass Spectrometry About 10 μL of the samples were measured using a Finnigan Surveyor Plus HPLC System coupled with a Triple Quadrupole TSQ Quantum Discovery Mass Spectrometer (Thermo Electron Corporation, Waltham, MA, USA) equipped with a Nucleosil 100-5 C18 EC 250/3 column (Macherey-Nagel, Düren, Germany). .. Aqueous formic acid (0.1%; eluent A) and acetonitrile containing 0.1% formic acid (eluent B) were used as eluents at a flow rate of 0.3 mL/min.

    Article Title: Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite
    Article Snippet: .. Liquid chromatography tandem mass spectrometry (LC–MS/MS) data were acquired on a Dionex UltiMate 3000 liquid chromatography system (Dionex, California, USA) coupled to an ABSciex 4000 QTRAP mass spectrometer (ABSciex, Concord, Canada). .. The liquid chromatography system was controlled by Chromeleon software (v6.80 SR9, Dionex), and chromatographic separation was achieved by injecting 10 μL onto a Gemini-NX C18 150 mm × 2 mm I.D., 3 μm 110 Å particle column (Phenomenex, Aschaffenburg, Germany) equipped with a pre-column Security Guard Gemini-NX C18 4 mm × 2 mm I.D. cartridge.

    Article Title: Pharmacogenetics Meets Metabolomics: Discovery of Tryptophan as a New Endogenous OCT2 Substrate Related to Metformin Disposition
    Article Snippet: .. Assay of tryptophan concentrations using liquid chromatography-tandem mass spectrometry Tryptophan concentrations in urine samples were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS; API 3000; Applied Biosystems, Foster City, CA). ..

    Article Title: Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor) Dose‐dependent acute liver injury with hypersensitivity features in humans due to a novel microsomal prostaglandin E synthase 1 inhibitor
    Article Snippet: .. Putative metabolite structures were identified using accurate mass liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry with positive electrospray ionization (LTQ Orbitrap XL, Thermo Scientific, Waltham, MA, USA) as well as metabolite retention time comparisons. ..

    Article Title: Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib
    Article Snippet: .. 2.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) For IMAC-enriched samples, reversed phase chromatography was performed on eluted peptides using a Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific). ..

    Reversed-phase Chromatography:

    Article Title: Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib
    Article Snippet: .. 2.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) For IMAC-enriched samples, reversed phase chromatography was performed on eluted peptides using a Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific). ..

    Liquid Chromatography:

    Article Title: Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite
    Article Snippet: .. Liquid chromatography tandem mass spectrometry (LC–MS/MS) data were acquired on a Dionex UltiMate 3000 liquid chromatography system (Dionex, California, USA) coupled to an ABSciex 4000 QTRAP mass spectrometer (ABSciex, Concord, Canada). .. The liquid chromatography system was controlled by Chromeleon software (v6.80 SR9, Dionex), and chromatographic separation was achieved by injecting 10 μL onto a Gemini-NX C18 150 mm × 2 mm I.D., 3 μm 110 Å particle column (Phenomenex, Aschaffenburg, Germany) equipped with a pre-column Security Guard Gemini-NX C18 4 mm × 2 mm I.D. cartridge.

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  • 92
    Thermo Fisher dgat2 transcripts
    13 C-Oleate Tracing Reveals a Critical Buffering Role for TG-Resident Unsaturated FAs (A) Effect of SCDi on total TG abundances as measured by LC-MS. (B) Effect of oleate pre-loading with or without DGAT shRNA on subsequent A498 cell survival (by Annexin-PI) during serum limitation and SCD inhibition. (C) Schematic of the experimental workflow. <t>DGAT2</t> knockout cells were serum-starved for 24 hr and then loaded for 24 hr with 10 μM [U 13 C]-oleate (C18:1) ± DGAT1 inhibitor (T863, 2 μM). The medium was then replaced and the tracer removed, and cells were subjected to a 48-hr washout. (D) TG labeling patterns after 24-hr loading with [U 13 C]-oleate with or without DGATi, where numbers of mono-unsaturated FA (MUFA) and FA carbons are indicated. 1×, 2×, and 3× indicate whether TGs have one, two, or three oleates (includes [ 13 C 18 ]-20:1) conjugated to their glycerol backbones. (E) BODIPY and DAPI staining directly after [U 13 C]-oleate loading with or without DGATi. (F) Labeling patterns as assessed by incorporation of the 13 C label in 18:1 and 20:1 FAs in TG, DG, PC, and PE species. (G) Model of the metabolic mechanism by which TGs alleviate the saturation of certain lipid classes (e.g., PCs) under conditions of unsaturated lipid deprivation by releasing stored oleate. Data are means of triplicate wells confirmed in independent experiments (A, B, and D) or means of three independent experiments each conducted in triplicate (F); error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗ p
    Dgat2 Transcripts, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher 293t cells
    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in <t>293T</t> cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 4378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher lc ms ms analysis all peptide
    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif <t>analysis</t> for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr <t>peptide</t> abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) <t>MS/MS</t> spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).
    Lc Ms Ms Analysis All Peptide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher analysis hplc separations
    Multivariate statistical analysis of <t>HPLC–PDA–MS</t> non-targeted profiles. a Conventional PCA scores plot inclusive of quality assurance samples. b Conventional PCA scores plot with quality assurance samples excluded. c Conventional PCA loadings plot with quality assurance samples excluded (please refer to Table S1 and S2 for metabolites associated with unique reference numbers). d Multiblock hierarchical (H)PCA super-scores plot. e Multiblock hierarchical (H)PCA block-scores plots based upon daylength condition. Natural, 10 h, 10 h + 3 h, refer to the following daylength condition descriptions, (1) natural long summer day (LD), ca. 18 h (natural LD), (2) 10 h artificial short day (SD), and (3) 10 h SD + 3 h night interruption (SD + NI), respectively
    Analysis Hplc Separations, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    13 C-Oleate Tracing Reveals a Critical Buffering Role for TG-Resident Unsaturated FAs (A) Effect of SCDi on total TG abundances as measured by LC-MS. (B) Effect of oleate pre-loading with or without DGAT shRNA on subsequent A498 cell survival (by Annexin-PI) during serum limitation and SCD inhibition. (C) Schematic of the experimental workflow. DGAT2 knockout cells were serum-starved for 24 hr and then loaded for 24 hr with 10 μM [U 13 C]-oleate (C18:1) ± DGAT1 inhibitor (T863, 2 μM). The medium was then replaced and the tracer removed, and cells were subjected to a 48-hr washout. (D) TG labeling patterns after 24-hr loading with [U 13 C]-oleate with or without DGATi, where numbers of mono-unsaturated FA (MUFA) and FA carbons are indicated. 1×, 2×, and 3× indicate whether TGs have one, two, or three oleates (includes [ 13 C 18 ]-20:1) conjugated to their glycerol backbones. (E) BODIPY and DAPI staining directly after [U 13 C]-oleate loading with or without DGATi. (F) Labeling patterns as assessed by incorporation of the 13 C label in 18:1 and 20:1 FAs in TG, DG, PC, and PE species. (G) Model of the metabolic mechanism by which TGs alleviate the saturation of certain lipid classes (e.g., PCs) under conditions of unsaturated lipid deprivation by releasing stored oleate. Data are means of triplicate wells confirmed in independent experiments (A, B, and D) or means of three independent experiments each conducted in triplicate (F); error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗ p

    Journal: Cell Reports

    Article Title: Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation

    doi: 10.1016/j.celrep.2018.08.015

    Figure Lengend Snippet: 13 C-Oleate Tracing Reveals a Critical Buffering Role for TG-Resident Unsaturated FAs (A) Effect of SCDi on total TG abundances as measured by LC-MS. (B) Effect of oleate pre-loading with or without DGAT shRNA on subsequent A498 cell survival (by Annexin-PI) during serum limitation and SCD inhibition. (C) Schematic of the experimental workflow. DGAT2 knockout cells were serum-starved for 24 hr and then loaded for 24 hr with 10 μM [U 13 C]-oleate (C18:1) ± DGAT1 inhibitor (T863, 2 μM). The medium was then replaced and the tracer removed, and cells were subjected to a 48-hr washout. (D) TG labeling patterns after 24-hr loading with [U 13 C]-oleate with or without DGATi, where numbers of mono-unsaturated FA (MUFA) and FA carbons are indicated. 1×, 2×, and 3× indicate whether TGs have one, two, or three oleates (includes [ 13 C 18 ]-20:1) conjugated to their glycerol backbones. (E) BODIPY and DAPI staining directly after [U 13 C]-oleate loading with or without DGATi. (F) Labeling patterns as assessed by incorporation of the 13 C label in 18:1 and 20:1 FAs in TG, DG, PC, and PE species. (G) Model of the metabolic mechanism by which TGs alleviate the saturation of certain lipid classes (e.g., PCs) under conditions of unsaturated lipid deprivation by releasing stored oleate. Data are means of triplicate wells confirmed in independent experiments (A, B, and D) or means of three independent experiments each conducted in triplicate (F); error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗ p

    Article Snippet: After selection with puromycin and G418, the knockdown of both DGAT1 and DGAT2 transcripts was confirmed by qRT-PCR (Taqman probes; ThermoFisher) DGAT2 knockout cell lines were generated by cloning sgRNA sequences 5′-TGTGCTCTACTTCACTTGGC-3′ and 5′-GTACATGAGGATGGCACTGC-3′ into the lentiviral vector lentiCrisprv2 (Addgene), generating lentivirus in HEK293T cells and transducing ccRCC cell lines with 25μl of un-concentrated supernatant.

    Techniques: Liquid Chromatography with Mass Spectroscopy, shRNA, Inhibition, Knock-Out, Labeling, Staining

    DGAT Loss Reduces Tumor Growth and Alters Lipid Composition In Vivo (A) Diagram of fatty acid and lipid synthesis and the influence of O 2 and exogenous lipid. (B) Growth curves for A498 xenograft tumors with induced (doxycycline chow) and un-induced (control chow) DGAT1 and DGAT2 shRNAs (hereafter called DGAT shRNA). (C) Tumor weights after necropsy. (D) Immunohistochemistry for cleaved caspase-3 and Ki67 in xenograft tumors collected on day 5 of treatment, with accompanying quantification. (E) Total TG abundance derived from summing individual TG species abundance after liquid chromatography-mass spectrometry (LC-MS) quantification. (F) TG species binned according to the number of fully saturated FA chains present and the abundance of each category summed and displayed as a ratio of doxycycline-treated versus control groups. All results are means of n = 10 tumors (2 tumors per mouse) per arm; error bars represent ± SD (B, D, and F) or ± SEM (C). Statistical significance by t test or ANOVA, as appropriate; ∗ p

    Journal: Cell Reports

    Article Title: Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation

    doi: 10.1016/j.celrep.2018.08.015

    Figure Lengend Snippet: DGAT Loss Reduces Tumor Growth and Alters Lipid Composition In Vivo (A) Diagram of fatty acid and lipid synthesis and the influence of O 2 and exogenous lipid. (B) Growth curves for A498 xenograft tumors with induced (doxycycline chow) and un-induced (control chow) DGAT1 and DGAT2 shRNAs (hereafter called DGAT shRNA). (C) Tumor weights after necropsy. (D) Immunohistochemistry for cleaved caspase-3 and Ki67 in xenograft tumors collected on day 5 of treatment, with accompanying quantification. (E) Total TG abundance derived from summing individual TG species abundance after liquid chromatography-mass spectrometry (LC-MS) quantification. (F) TG species binned according to the number of fully saturated FA chains present and the abundance of each category summed and displayed as a ratio of doxycycline-treated versus control groups. All results are means of n = 10 tumors (2 tumors per mouse) per arm; error bars represent ± SD (B, D, and F) or ± SEM (C). Statistical significance by t test or ANOVA, as appropriate; ∗ p

    Article Snippet: After selection with puromycin and G418, the knockdown of both DGAT1 and DGAT2 transcripts was confirmed by qRT-PCR (Taqman probes; ThermoFisher) DGAT2 knockout cell lines were generated by cloning sgRNA sequences 5′-TGTGCTCTACTTCACTTGGC-3′ and 5′-GTACATGAGGATGGCACTGC-3′ into the lentiviral vector lentiCrisprv2 (Addgene), generating lentivirus in HEK293T cells and transducing ccRCC cell lines with 25μl of un-concentrated supernatant.

    Techniques: In Vivo, shRNA, Immunohistochemistry, Derivative Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    TGs Promote Cell Viability in Low O 2 and Serum by Absorbing FA Saturation (A) Viability of A498 cells expressing inducible shRNA against DGAT1 and DGAT2 mRNAs ( DGAT shRNA), assessed after 72 hr under the indicated conditions (hypoxia = 0.5% O 2 ; serum deprivation = low serum, 0.5% fetal bovine serum [FBS]) by Annexin-propidium iodide (PI) flow cytometry assay. (B) Viability of cells expressing inducible DGAT shRNAs after 72 hr under the indicated conditions (SCDi, 1 μM CAY10566) by Annexin-PI assay using flow cytometry. (C) Volcano plot showing fold change and significance of alterations in the lipidome of A498 cells cultured in low (0.5%) versus high (5%) serum. Lipids with ≥ 1.5 fold change and p ≤ 0.05 are displayed in color to denote lipid class. (D) Changes in FA composition or saturation of TGs, calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values are normalized to control conditions (5% serum). (E) Lipid class-specific saturation indices (defined by (palmitate + stearate) / oleate) for A498 cells cultured under hypoxic (0.5% O 2 ) versus normoxic conditions (both in low serum). (F) As (E) but with pharmacological SCD inhibition (1 μM CAY10566) instead of hypoxia. (G) Effect of serum deprivation and DGAT shRNA on total TG abundances. (H) Changes in FA makeup of TGs following DGAT knockdown; values were calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values were normalized to the control condition (vehicle [Veh] treatment). (I) TG saturation indices for the indicated conditions. Values are relative to normoxic untreated cells. (J) As (G) but with pharmacological SCD inhibition (1 μM CAY10566). Values are relative to the untreated vehicle control. Data are means of 3 (A, B, and D–J) or 5 (C) replicate wells and were confirmed in independent experiments; error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗∗ p

    Journal: Cell Reports

    Article Title: Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation

    doi: 10.1016/j.celrep.2018.08.015

    Figure Lengend Snippet: TGs Promote Cell Viability in Low O 2 and Serum by Absorbing FA Saturation (A) Viability of A498 cells expressing inducible shRNA against DGAT1 and DGAT2 mRNAs ( DGAT shRNA), assessed after 72 hr under the indicated conditions (hypoxia = 0.5% O 2 ; serum deprivation = low serum, 0.5% fetal bovine serum [FBS]) by Annexin-propidium iodide (PI) flow cytometry assay. (B) Viability of cells expressing inducible DGAT shRNAs after 72 hr under the indicated conditions (SCDi, 1 μM CAY10566) by Annexin-PI assay using flow cytometry. (C) Volcano plot showing fold change and significance of alterations in the lipidome of A498 cells cultured in low (0.5%) versus high (5%) serum. Lipids with ≥ 1.5 fold change and p ≤ 0.05 are displayed in color to denote lipid class. (D) Changes in FA composition or saturation of TGs, calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values are normalized to control conditions (5% serum). (E) Lipid class-specific saturation indices (defined by (palmitate + stearate) / oleate) for A498 cells cultured under hypoxic (0.5% O 2 ) versus normoxic conditions (both in low serum). (F) As (E) but with pharmacological SCD inhibition (1 μM CAY10566) instead of hypoxia. (G) Effect of serum deprivation and DGAT shRNA on total TG abundances. (H) Changes in FA makeup of TGs following DGAT knockdown; values were calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values were normalized to the control condition (vehicle [Veh] treatment). (I) TG saturation indices for the indicated conditions. Values are relative to normoxic untreated cells. (J) As (G) but with pharmacological SCD inhibition (1 μM CAY10566). Values are relative to the untreated vehicle control. Data are means of 3 (A, B, and D–J) or 5 (C) replicate wells and were confirmed in independent experiments; error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗∗ p

    Article Snippet: After selection with puromycin and G418, the knockdown of both DGAT1 and DGAT2 transcripts was confirmed by qRT-PCR (Taqman probes; ThermoFisher) DGAT2 knockout cell lines were generated by cloning sgRNA sequences 5′-TGTGCTCTACTTCACTTGGC-3′ and 5′-GTACATGAGGATGGCACTGC-3′ into the lentiviral vector lentiCrisprv2 (Addgene), generating lentivirus in HEK293T cells and transducing ccRCC cell lines with 25μl of un-concentrated supernatant.

    Techniques: Expressing, shRNA, Flow Cytometry, Cytometry, Cell Culture, Inhibition

    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Cell Culture

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: Kinase inhibition GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific).

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging

    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Sequencing, Mass Spectrometry

    Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Isolation

    Multivariate statistical analysis of HPLC–PDA–MS non-targeted profiles. a Conventional PCA scores plot inclusive of quality assurance samples. b Conventional PCA scores plot with quality assurance samples excluded. c Conventional PCA loadings plot with quality assurance samples excluded (please refer to Table S1 and S2 for metabolites associated with unique reference numbers). d Multiblock hierarchical (H)PCA super-scores plot. e Multiblock hierarchical (H)PCA block-scores plots based upon daylength condition. Natural, 10 h, 10 h + 3 h, refer to the following daylength condition descriptions, (1) natural long summer day (LD), ca. 18 h (natural LD), (2) 10 h artificial short day (SD), and (3) 10 h SD + 3 h night interruption (SD + NI), respectively

    Journal: Metabolomics

    Article Title: Application of HPLC–PDA–MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit

    doi: 10.1007/s11306-018-1462-5

    Figure Lengend Snippet: Multivariate statistical analysis of HPLC–PDA–MS non-targeted profiles. a Conventional PCA scores plot inclusive of quality assurance samples. b Conventional PCA scores plot with quality assurance samples excluded. c Conventional PCA loadings plot with quality assurance samples excluded (please refer to Table S1 and S2 for metabolites associated with unique reference numbers). d Multiblock hierarchical (H)PCA super-scores plot. e Multiblock hierarchical (H)PCA block-scores plots based upon daylength condition. Natural, 10 h, 10 h + 3 h, refer to the following daylength condition descriptions, (1) natural long summer day (LD), ca. 18 h (natural LD), (2) 10 h artificial short day (SD), and (3) 10 h SD + 3 h night interruption (SD + NI), respectively

    Article Snippet: Untargeted HPLC–PDA–MS analysis HPLC separations were performed with a Thermo Accela 600 HPLC system coupled with an Accela PDA detector (Thermo-Fisher Ltd. UK).

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry, Blocking Assay