nano liquid chromatography tandem mass spectrometry  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92

    Structured Review

    Thermo Fisher nano liquid chromatography tandem mass spectrometry
    Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma <t>cruzi</t> cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by <t>nano-liquid</t> chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.
    Nano Liquid Chromatography Tandem Mass Spectrometry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano liquid chromatography tandem mass spectrometry/product/Thermo Fisher
    Average 92 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    nano liquid chromatography tandem mass spectrometry - by Bioz Stars, 2020-07
    92/100 stars

    Images

    1) Product Images from "Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation"

    Article Title: Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation

    Journal: Genes

    doi: 10.3390/genes9080413

    Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma cruzi cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.
    Figure Legend Snippet: Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma cruzi cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.

    Techniques Used: Mass Spectrometry, Protein Extraction, Liquid Chromatography, Functional Assay, Microscopy, Staining

    2) Product Images from "The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells"

    Article Title: The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2014.03.002

    Lysine 528 on PARP1 is methylated by SMYD2. (A) The nano-LC-MS/MS spectrum of the specific peptide after in vitro methyltransferase assay (upper panel) and the theoretical value table of MS fragments (lower panel). The values observed were indicated in
    Figure Legend Snippet: Lysine 528 on PARP1 is methylated by SMYD2. (A) The nano-LC-MS/MS spectrum of the specific peptide after in vitro methyltransferase assay (upper panel) and the theoretical value table of MS fragments (lower panel). The values observed were indicated in

    Techniques Used: Methylation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro

    Related Articles

    Generated:

    Article Title: Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris
    Article Snippet: .. Tryptic peptides generated as described in the preceding section were analyzed through nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS) using an Ultimate 3000 nLC system (Dionex) connected to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific). ..

    Chromatography:

    Article Title: Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation
    Article Snippet: .. Mass Spectrometry-Based Analysis of T. cruzi Tryptic Peptides The peptides were separated by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an EASY-Spray PepMap® 50 cm × 75 μm C18 Column using an Easy nLC1000 nanoflow system (Thermo Fisher Scientific, Waltham, MA, USA). .. The high-performance liquid chromatography (HPLC) gradient was 5–25% solvent B (A 0.1% formic acid; B 100% acetonitrile (ACN), 0.1% formic acid) for 90 min at a flow of 300 nL/min.

    Article Title: Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris
    Article Snippet: .. Tryptic peptides generated as described in the preceding section were analyzed through nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS) using an Ultimate 3000 nLC system (Dionex) connected to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific). ..

    Article Title: Tri-methylation of ATF7IP by G9a/GLP recruits the chromodomain protein MPP8
    Article Snippet: .. The digests were applied to nano-liquid chromatography–tandem mass spectrometry using a Q Exactive mass spectrometer (Thermo Fisher Scientific). .. The peptide mixtures (2 μL) were separated by nano-ESI spray column (75 μm [ID] × 100 mm [L], NTCC analytical column C18, 3 μm, Nikkyo Technos) with a linear gradient of 0–35% buffer B (acetonitrile with 0.1% (v/v) formic acid) in buffer A (MilliQ water with 0.1% (v/v) formic acid) at a flow rate of 300 nL/min over 10 min (EAST-nLC 1000; Thermo Fisher Scientific).

    Article Title: Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation
    Article Snippet: .. The sample was analysed by nano‐liquid chromatography‐tandem mass spectrometry (nano‐LC‐MS/MS) in a Thermo Scientific Q Exactive Mass Spectrometer, (Waltham, Massachusetts, USA) ionized by electrospray with EASY‐SPRAY. ..

    Article Title: A Novel SRP Recognition Sequence in the Homeostatic Control Region of Heat Shock Transcription Factor σ32
    Article Snippet: .. Then digest was analyzed by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using Q Exactive mass spectrometer (Thermo Fisher Scientific). .. The peptides were separated using nano ESI spray column (75 μm [ID] × 100 mm [L], NTCC analytical column C18, 3 μm, Nikkyo Technos) with a linear gradient of 0%-35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min over 10 min (Easy nLC; Thermo Fisher Scientific).

    Article Title: Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering
    Article Snippet: .. Peptide digests were analyzed by nano Liquid Chromatography-tandem mass spectrometry (LC-MS/MS), on a Thermo Fisher LTQ OrbitrapVelos connected to a Waters Acquity UPLC system (Waters Corp., Milford, MA), using a 90 minute gradient. .. Protein identification was performed with Proteome Discoverer 1.3 using the Sequest search engine.

    Article Title: The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells
    Article Snippet: .. The reaction mixture of in vitro methyltransferase assay was analyzed by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using LCQ Deca XP plus (Thermo Fisher Scientific, San Jose, CA). .. The peptides were separated using nano ESI spray column (100 μm [ID] × 50 mm [L]) packed with a reversed-phase material (Inertsil ODS-3,3 μm; GL Sciences, Tokyo, Japan) at a flow rate 200 nl/min.

    Article Title: Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron
    Article Snippet: .. Samples were analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) on an Ultimate 3000 system (Dionex, Amsterdam, The Netherlands) interfaced on-line with an LTQ-Orbitrap-XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). ..

    Mass Spectrometry:

    Article Title: Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation
    Article Snippet: .. Mass Spectrometry-Based Analysis of T. cruzi Tryptic Peptides The peptides were separated by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an EASY-Spray PepMap® 50 cm × 75 μm C18 Column using an Easy nLC1000 nanoflow system (Thermo Fisher Scientific, Waltham, MA, USA). .. The high-performance liquid chromatography (HPLC) gradient was 5–25% solvent B (A 0.1% formic acid; B 100% acetonitrile (ACN), 0.1% formic acid) for 90 min at a flow of 300 nL/min.

    Article Title: Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris
    Article Snippet: .. Tryptic peptides generated as described in the preceding section were analyzed through nano liquid chromatography–tandem mass spectrometry (nLC-MS/MS) using an Ultimate 3000 nLC system (Dionex) connected to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific). ..

    Article Title: Tri-methylation of ATF7IP by G9a/GLP recruits the chromodomain protein MPP8
    Article Snippet: .. The digests were applied to nano-liquid chromatography–tandem mass spectrometry using a Q Exactive mass spectrometer (Thermo Fisher Scientific). .. The peptide mixtures (2 μL) were separated by nano-ESI spray column (75 μm [ID] × 100 mm [L], NTCC analytical column C18, 3 μm, Nikkyo Technos) with a linear gradient of 0–35% buffer B (acetonitrile with 0.1% (v/v) formic acid) in buffer A (MilliQ water with 0.1% (v/v) formic acid) at a flow rate of 300 nL/min over 10 min (EAST-nLC 1000; Thermo Fisher Scientific).

    Article Title: Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation
    Article Snippet: .. The sample was analysed by nano‐liquid chromatography‐tandem mass spectrometry (nano‐LC‐MS/MS) in a Thermo Scientific Q Exactive Mass Spectrometer, (Waltham, Massachusetts, USA) ionized by electrospray with EASY‐SPRAY. ..

    Article Title: A Novel SRP Recognition Sequence in the Homeostatic Control Region of Heat Shock Transcription Factor σ32
    Article Snippet: .. Then digest was analyzed by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using Q Exactive mass spectrometer (Thermo Fisher Scientific). .. The peptides were separated using nano ESI spray column (75 μm [ID] × 100 mm [L], NTCC analytical column C18, 3 μm, Nikkyo Technos) with a linear gradient of 0%-35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min over 10 min (Easy nLC; Thermo Fisher Scientific).

    Article Title: Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering
    Article Snippet: .. Peptide digests were analyzed by nano Liquid Chromatography-tandem mass spectrometry (LC-MS/MS), on a Thermo Fisher LTQ OrbitrapVelos connected to a Waters Acquity UPLC system (Waters Corp., Milford, MA), using a 90 minute gradient. .. Protein identification was performed with Proteome Discoverer 1.3 using the Sequest search engine.

    Article Title: The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells
    Article Snippet: .. The reaction mixture of in vitro methyltransferase assay was analyzed by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using LCQ Deca XP plus (Thermo Fisher Scientific, San Jose, CA). .. The peptides were separated using nano ESI spray column (100 μm [ID] × 50 mm [L]) packed with a reversed-phase material (Inertsil ODS-3,3 μm; GL Sciences, Tokyo, Japan) at a flow rate 200 nl/min.

    Article Title: Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron
    Article Snippet: .. Samples were analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) on an Ultimate 3000 system (Dionex, Amsterdam, The Netherlands) interfaced on-line with an LTQ-Orbitrap-XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). ..

    In Vitro:

    Article Title: The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells
    Article Snippet: .. The reaction mixture of in vitro methyltransferase assay was analyzed by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using LCQ Deca XP plus (Thermo Fisher Scientific, San Jose, CA). .. The peptides were separated using nano ESI spray column (100 μm [ID] × 50 mm [L]) packed with a reversed-phase material (Inertsil ODS-3,3 μm; GL Sciences, Tokyo, Japan) at a flow rate 200 nl/min.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Thermo Fisher nano lc ms ms
    TLR4 interacts with myosin-9. (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by <t>nano-LC/MS/MS</t> on an <t>LCQ</t> Deca XP Plus ion trap mass spectrometer.
    Nano Lc Ms Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano lc ms ms/product/Thermo Fisher
    Average 93 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    nano lc ms ms - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    92
    Thermo Fisher nano liquid chromatography tandem mass spectrometry
    Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma <t>cruzi</t> cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by <t>nano-liquid</t> chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.
    Nano Liquid Chromatography Tandem Mass Spectrometry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano liquid chromatography tandem mass spectrometry/product/Thermo Fisher
    Average 92 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    nano liquid chromatography tandem mass spectrometry - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    85
    Thermo Fisher nano flow capillary ultra high performance lc system
    pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution <t>nano-UPLC-MS</t> after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).
    Nano Flow Capillary Ultra High Performance Lc System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano flow capillary ultra high performance lc system/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nano flow capillary ultra high performance lc system - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    Image Search Results


    TLR4 interacts with myosin-9. (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.

    Journal: PLoS ONE

    Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in ?-Granules Trafficking

    doi: 10.1371/journal.pone.0085833

    Figure Lengend Snippet: TLR4 interacts with myosin-9. (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.

    Article Snippet: The tryptic peptides were then analyzed by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to an Agilent 1100 HPLC (Agilent Technologies, Inc., San Jose, CA, USA).

    Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

    On exposure to the MPO-H 2 O 2 -NaNO 2 system, Tyr-192 is the major nitration target in apoA-I, but Tyr-18 is the major target when apoA-I is associated with HDL. Lipid-free apoA-I (10 μ m ) ( A and B ) or HDL 3 (0.5 mg/ml, ∼12.5 μ m apoA-I)

    Journal: The Journal of Biological Chemistry

    Article Title: Myeloperoxidase Targets Apolipoprotein A-I, the Major High Density Lipoprotein Protein, for Site-Specific Oxidation in Human Atherosclerotic Lesions *

    doi: 10.1074/jbc.M111.337345

    Figure Lengend Snippet: On exposure to the MPO-H 2 O 2 -NaNO 2 system, Tyr-192 is the major nitration target in apoA-I, but Tyr-18 is the major target when apoA-I is associated with HDL. Lipid-free apoA-I (10 μ m ) ( A and B ) or HDL 3 (0.5 mg/ml, ∼12.5 μ m apoA-I)

    Article Snippet: After digesting HDL with trypsin or Glu-C, we used nano-LC-MS/MS and SRM with oxidized [15 N]apoA-I to quantify peptides of apoA-I that contained tyrosine.

    Techniques: Nitration

    Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma cruzi cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.

    Journal: Genes

    Article Title: Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation

    doi: 10.3390/genes9080413

    Figure Lengend Snippet: Mass spectrometry-based proteomics approach to assess the regulation of proteins during transitioning from exponential to stationary phase in Trypanosoma cruzi cells. ( A ) Proteomics workflow for identification and quantification of regulated proteins in T. cruzi during growth phase transitioning. T. cruzi epimastigote cells were taken during the exponential and stationary phase before protein extraction and tryptic digestion. Tryptic peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. After data and bioinformatic analysis combined with functional studies, this approach made possible the assessment of protein regulation during T. cruzi exponential to stationary phase transitioning. ( B ) Epimastigotes growth curve presenting typical exponential and stationary phases with physiological differences. Optic microscopy representative images show the different shape of the exponential and stationary phases of the parasite. ( C ) Microscopic analysis of T. cruzi epimastigotes at the exponential and stationary phases using phase-contrast microscope and DAPI staining. DIC: differential interference contrast. Scale bar: 4 μm.

    Article Snippet: Mass Spectrometry-Based Analysis of T. cruzi Tryptic Peptides The peptides were separated by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an EASY-Spray PepMap® 50 cm × 75 μm C18 Column using an Easy nLC1000 nanoflow system (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Mass Spectrometry, Protein Extraction, Liquid Chromatography, Functional Assay, Microscopy, Staining

    pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).

    Journal: Nature chemical biology

    Article Title: A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    doi: 10.1038/nchembio.1259

    Figure Lengend Snippet: pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).

    Article Snippet: High-resolution nano-UPLC-MS analysis of pHis proteins and peptides LC-MS and MS/MS analyses of pHis samples were performed on high-resolution, high-mass-accuracy, reversed-phase nano-UPLC-MS platforms, consisting of either a nano-flow capillary ultra high performance LC system (Nano Ultra 2D Plus, Eksigent) coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher Scientific) outfitted with a Triversa NanoMate ion source robot (Advion), or an Easy nLC Ultra 1000 nano-UPLC system (ThermoFisher Scientific) coupled to a Velos Pro-Orbi Elite hybrid mass spectrometer (ThermoFisher Scientific) equipped with a Flex Ion source (Proxeon Biosystems).

    Techniques: Western Blot, Staining, Mass Spectrometry, Sequencing, Chick Chorioallantoic Membrane Assay, Mass Measurement, Inhibition, De-Phosphorylation Assay

    Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).

    Journal: Nature chemical biology

    Article Title: A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    doi: 10.1038/nchembio.1259

    Figure Lengend Snippet: Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).

    Article Snippet: High-resolution nano-UPLC-MS analysis of pHis proteins and peptides LC-MS and MS/MS analyses of pHis samples were performed on high-resolution, high-mass-accuracy, reversed-phase nano-UPLC-MS platforms, consisting of either a nano-flow capillary ultra high performance LC system (Nano Ultra 2D Plus, Eksigent) coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher Scientific) outfitted with a Triversa NanoMate ion source robot (Advion), or an Easy nLC Ultra 1000 nano-UPLC system (ThermoFisher Scientific) coupled to a Velos Pro-Orbi Elite hybrid mass spectrometer (ThermoFisher Scientific) equipped with a Flex Ion source (Proxeon Biosystems).

    Techniques: Western Blot, Expressing, Purification, Mass Spectrometry, Sequencing, Mass Measurement, Dot Blot, Concentration Assay