chromatography analysis  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Liquid Chromatography
    Description:
    With the advent of new interfacing technology the benefits of LC MS liquid chromatography with mass spectrometry are being realized by the growth in applications of this technique in both the chemical and life sciences Topics covered include Limitations of the component techniques when used in isolation and how a combination of the two allows these to be overcome Descriptions of the various approaches including thermospray electrospray and atmospheric pressure chemical ionization which are used to interface the two techniques along with the advantages and disadvantages of each system Illustrative examples of the applications of LC MS including the molecular weight and structure determinations of both biopolymers and low molecular compounds
    Catalog Number:
    Z702080
    Price:
    None
    Buy from Supplier


    Structured Review

    Millipore chromatography analysis
    Liquid Chromatography
    With the advent of new interfacing technology the benefits of LC MS liquid chromatography with mass spectrometry are being realized by the growth in applications of this technique in both the chemical and life sciences Topics covered include Limitations of the component techniques when used in isolation and how a combination of the two allows these to be overcome Descriptions of the various approaches including thermospray electrospray and atmospheric pressure chemical ionization which are used to interface the two techniques along with the advantages and disadvantages of each system Illustrative examples of the applications of LC MS including the molecular weight and structure determinations of both biopolymers and low molecular compounds
    https://www.bioz.com/result/chromatography analysis/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chromatography analysis - by Bioz Stars, 2021-07
    99/100 stars

    Images

    Related Articles

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar
    Article Snippet: The mucins were purified on an in-house prepared one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS Ag-PAGE) and electroblotted to a PVDF membrane (ImmobilonPSQ) using a semidry transfer method, as previously described [ ]. .. O-Glycan Analysis by LC-MS Mucin bands on the PVDF membranes were visualized by Alcian blue (Sigma–Aldrich, St. Louis, MO, USA) staining. ..

    Article Title: Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios
    Article Snippet: .. Detection of Indigoidine Production by Leisingera sp. JC1 via LC–MS All HPLC grade solvents and reagents were purchased from Sigma-Aldrich. .. LC–MS data were collected on an Agilent ESI single quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system with a G1311 quaternary pump, G1322 degasser, and a G1315 diode array detector (Agilent Technologies, Santa Clara, CA).

    Article Title: Nonclinical Evaluation of PF-06438179: A Potential Biosimilar to Remicade® (Infliximab)
    Article Snippet: .. Peptide Mapping Using Liquid Chromatography/Mass Spectrometry (LC/MS) PF-06438179, infliximab-EU, and infliximab-US samples were denatured in 8-M guanidine HCl (Sigma-Aldrich), then reduced with 1,4-dithio-dl -threitol (DTT; Sigma-Aldrich) at 40 °C for 1 h and alkylated with iodoacetic acid, sodium salt (Sigma-Aldrich) at room temperature for 1 h. The reduced/alkylated samples were desalted using a fast desalting 10/100 GL column (GE Healthcare Life Sciences) into 50 mM tris buffer pH 8.2, and digested with trypsin (Roche) at 37 °C overnight. .. The resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (3.5 μm, 2.1 × 250 mm; Waters BioSuite™) at 35 °C with an acetonitrile gradient containing trifluroacetic acid (Thermo Scientific).

    Staining:

    Article Title: Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar
    Article Snippet: The mucins were purified on an in-house prepared one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS Ag-PAGE) and electroblotted to a PVDF membrane (ImmobilonPSQ) using a semidry transfer method, as previously described [ ]. .. O-Glycan Analysis by LC-MS Mucin bands on the PVDF membranes were visualized by Alcian blue (Sigma–Aldrich, St. Louis, MO, USA) staining. ..

    Chromatography:

    Article Title: Diabetic Endothelial Cells Differentiated From Patient iPSCs Show Dysregulated Glycine Homeostasis and Senescence Associated Phenotypes
    Article Snippet: .. The amino acid extracts were derivatized with 3 M Hydrochloric acid in methanol or butanol (Sigma-Aldrich, United States), dried, and reconstituted in methanol for analysis in Liquid chromatography–mass spectrometry. .. Methods of amino acid analysis were modified from .

    Article Title: Nonclinical Evaluation of PF-06438179: A Potential Biosimilar to Remicade® (Infliximab)
    Article Snippet: .. Peptide Mapping Using Liquid Chromatography/Mass Spectrometry (LC/MS) PF-06438179, infliximab-EU, and infliximab-US samples were denatured in 8-M guanidine HCl (Sigma-Aldrich), then reduced with 1,4-dithio-dl -threitol (DTT; Sigma-Aldrich) at 40 °C for 1 h and alkylated with iodoacetic acid, sodium salt (Sigma-Aldrich) at room temperature for 1 h. The reduced/alkylated samples were desalted using a fast desalting 10/100 GL column (GE Healthcare Life Sciences) into 50 mM tris buffer pH 8.2, and digested with trypsin (Roche) at 37 °C overnight. .. The resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (3.5 μm, 2.1 × 250 mm; Waters BioSuite™) at 35 °C with an acetonitrile gradient containing trifluroacetic acid (Thermo Scientific).

    Article Title: The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability
    Article Snippet: To estimate pulmonary vascular permeability, BAL supernatant was analyzed for IgM using an enzyme immunoassay (Immunology Consultant Laboratory). .. Electrospray ionization–liquid chromatography–tandem mass spectrometry Plasma samples were processed by filtering through a Millipore 10,000 molecular weight cutoff centrifugal filter and then by washing with 40 μl of 1 mM HCl. .. PGP peptides in plasma and BAL were quantified using two different devices: For BAL, a MDS Sciex (Applied Biosystems) API-4000 triple quadrupole mass spectrometer equipped with a Shimadzu high-performance liquid chromatography (HPLC) and a 2.0 × 150–mm Jupiter 4μ Proteo column (Phenomenex) was used.

    Purification:

    Article Title: Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase
    Article Snippet: The demethylated peptides were detected by immuno-blotting with the affinity purified anti-monomethylated K142 antibody or the anti-DNMT1 antibody for total peptides. .. Immuno-affinity purification and mass spectrometry analysis Cell lysates from twenty dishes (150 mm × 25 mm) of HCT116 cells containing the knock-in Flag-DNMT1 or equal dishes of control HCT116 cells were employed for immuno-affinity purification using 1 ml of the anti-Flag M2 affinity gel (Sigma, A2220) , . .. The proteins in the Flag-DNMT1 immunocomplexes were separated on an SDS-PAGE gel.

    Mass Spectrometry:

    Article Title: Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase
    Article Snippet: The demethylated peptides were detected by immuno-blotting with the affinity purified anti-monomethylated K142 antibody or the anti-DNMT1 antibody for total peptides. .. Immuno-affinity purification and mass spectrometry analysis Cell lysates from twenty dishes (150 mm × 25 mm) of HCT116 cells containing the knock-in Flag-DNMT1 or equal dishes of control HCT116 cells were employed for immuno-affinity purification using 1 ml of the anti-Flag M2 affinity gel (Sigma, A2220) , . .. The proteins in the Flag-DNMT1 immunocomplexes were separated on an SDS-PAGE gel.

    Article Title: The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability
    Article Snippet: To estimate pulmonary vascular permeability, BAL supernatant was analyzed for IgM using an enzyme immunoassay (Immunology Consultant Laboratory). .. Electrospray ionization–liquid chromatography–tandem mass spectrometry Plasma samples were processed by filtering through a Millipore 10,000 molecular weight cutoff centrifugal filter and then by washing with 40 μl of 1 mM HCl. .. PGP peptides in plasma and BAL were quantified using two different devices: For BAL, a MDS Sciex (Applied Biosystems) API-4000 triple quadrupole mass spectrometer equipped with a Shimadzu high-performance liquid chromatography (HPLC) and a 2.0 × 150–mm Jupiter 4μ Proteo column (Phenomenex) was used.

    Knock-In:

    Article Title: Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase
    Article Snippet: The demethylated peptides were detected by immuno-blotting with the affinity purified anti-monomethylated K142 antibody or the anti-DNMT1 antibody for total peptides. .. Immuno-affinity purification and mass spectrometry analysis Cell lysates from twenty dishes (150 mm × 25 mm) of HCT116 cells containing the knock-in Flag-DNMT1 or equal dishes of control HCT116 cells were employed for immuno-affinity purification using 1 ml of the anti-Flag M2 affinity gel (Sigma, A2220) , . .. The proteins in the Flag-DNMT1 immunocomplexes were separated on an SDS-PAGE gel.

    High Performance Liquid Chromatography:

    Article Title: Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios
    Article Snippet: .. Detection of Indigoidine Production by Leisingera sp. JC1 via LC–MS All HPLC grade solvents and reagents were purchased from Sigma-Aldrich. .. LC–MS data were collected on an Agilent ESI single quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system with a G1311 quaternary pump, G1322 degasser, and a G1315 diode array detector (Agilent Technologies, Santa Clara, CA).

    Article Title: Nitrite and Nitrate Levels in Follicular Fluid From Human Oocyte Donors Are Related to Ovarian Response and Embryo Quality
    Article Snippet: .. High-performance liquid chromatography standards were prepared in Milli-Q water using reagent grade sodium nitrite and sodium nitrate (Sigma-Aldrich Química S.A., Madrid, Spain). .. Both standards were prepared at a concentration of 1 mM, and serial dilutions from 100 to 0.1 μM were used to obtain the calibration curve for the analysis.

    Molecular Weight:

    Article Title: The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability
    Article Snippet: To estimate pulmonary vascular permeability, BAL supernatant was analyzed for IgM using an enzyme immunoassay (Immunology Consultant Laboratory). .. Electrospray ionization–liquid chromatography–tandem mass spectrometry Plasma samples were processed by filtering through a Millipore 10,000 molecular weight cutoff centrifugal filter and then by washing with 40 μl of 1 mM HCl. .. PGP peptides in plasma and BAL were quantified using two different devices: For BAL, a MDS Sciex (Applied Biosystems) API-4000 triple quadrupole mass spectrometer equipped with a Shimadzu high-performance liquid chromatography (HPLC) and a 2.0 × 150–mm Jupiter 4μ Proteo column (Phenomenex) was used.

    Liquid Chromatography:

    Article Title: Spatial variability and depuration of tetrodotoxin in the bivalve Paphies australis from New Zealand
    Article Snippet: Samples were then centrifuged (17,000×g, 1 min) and the supernatant subjected to the graphitised carbon solid phase extraction (SPE) method as described in using Supelclean ENVI-Carb 250 mg/3 mL cartridges (Sigma-Aldrich, MO, USA). .. Tetrodotoxin was analysed and quantified by liquid chromatography tandem-mass spectrometry analysis as described by . ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore tlc analysis
    Solution NMR and 31 P and 2 H solid-state NMR analysis. Thin-layer chromatography analysis of <t>Phosphoinositides</t> mix (PIPs). ( A ) REM-CA folds in alpha helix in hydrophobic environment. 2D 1 H- 15 N correlation spectra of REM-CA recorded at 800MHz using the SOFAST-HMQC pulse sequence. All spectra were recorded at 25°C with 1 mM of REMCA-WT in H 2 O/D 2 O (90/10) (red), Dodecylphosphocholine-d38, DPC (blue) and trifluoroethanol-d2, TFE (grey). ( B ) Primulin-stained high-performance thin layer chromatography <t>(HP-TLC)</t> plate of the Phosphoinositides mix (PIPs), reference P6023 SIGMA, along with authentic standards: Phosphatidylserine (PS) Phosphatidylinositol (PI), Phosphatidylinositol-4-phosphate (PI4P) and Phosphatidylinositol-4,5-bisphosphate PI(4,5)P 2 ). ( C ) Histogram representation of relative amounts of each lipid species present in the PIPs quantified by densitometry scanning. Bars indicate SEM, n = 3. ( D ) Temperature effect on REM-CA containing liposomes in the range −8°C to 31°C. Heating curves and corresponding NMR lineshape do not show significant changes. ( E ) 31 P NMR spectra of deuterated 1,2-dimyristoyl-d54- sn -3-phosphocholine (DMPC-d54) membrane in the absence or presence of REM-CA. DOI: http://dx.doi.org/10.7554/eLife.26404.010
    Tlc Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tlc analysis/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tlc analysis - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    97
    Millipore maldi tof ms analysis
    Time-dependent mass spectrometry analyses of CrGSNOR1 treated with Biotin-maleimide . Recombinant CrGSNOR1 was incubated in the presence of 1 mM Biotin-maleimide. At indicated time points, protein samples were withdrawn and analyzed by <t>MALDI-TOF</t> MS to assess the number of alkylated cysteines. For each alkylated cysteine, the molecular mass of CrGSNOR1 is shifted by +451 Da compared to the native protein (41473.4 Da). Peaks highlighted by an asterisk correspond to the protein-matrix (sinapinic acid) adduct. The y-axis is equal for all mass spectra acquired at times 0, 30, 60, and 90 min, and only indicated in the bottom spectrum.
    Maldi Tof Ms Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/maldi tof ms analysis/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    maldi tof ms analysis - by Bioz Stars, 2021-07
    97/100 stars
      Buy from Supplier

    97
    Millipore proteins lysostaphin
    Förster Resonance Energy Transfer assays for peptidoglycan hydrolases. FRET assays were performed with the synthetic peptide Dabcyl-Ala-Glu-Lys-Ala-Gly 5 -EDANS in 20 mM Tris pH 8.0 supplemented with 2 mM CaCl 2 , and measured at 340/490 nm. A) The effect of varying concentration of PlySs2 (0–40 μM) in the presence of 5 μM peptide. B) The effect of varying concentration of peptide (0–10 μM) in the presence of 5 μM PlySs2. C) The kinetics of <t>lysostaphin,</t> ClyS and PlySs2 (5 μM) in the presence of 5 μM peptide. D) The kinetics of ClyS under a different ClyS:peptide ratio. E) The V 0 of PlySs2 at different concentrations of peptide (substrate) was calculated based on the data points from Fig 4B, and plotted in a V 0 /[S 0 ] graph, with a two degree polynomial function used to fit the data points. F) A Lineweaver-Burk plot of 1/V 0 vs 1/[S 0 ], with one outlier removed for clarity. The graph was used for calculating the k cat and K m . All experiments were conducted in triplicates, at least three times, and representative figures are shown.
    Proteins Lysostaphin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteins lysostaphin/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteins lysostaphin - by Bioz Stars, 2021-07
    97/100 stars
      Buy from Supplier

    99
    Millipore mass spectrometry analysis cell lysates
    Regulation of DNMT1 protein stability by DCAF5. a Isolated DNMT1 protein complexes contain DDB1, DCAF5, and L3MBTL3 proteins (see Supplementary Table 1 for additional information). The Flag-knock-in DNMT1 protein complexes were isolated from HCT116-Flag-DNMT1 cells by anti-Flag M2 antibody affinity resins or from control HCT116 cells without the Flag-DNMT1. The proteins associated with the isolated Flag-DNMT1 complexes were separated in protein gel, excised, trypsinized, and derivative peptides were fractionated by nano-liter liquid chromatography and identified by <t>mass</t> <t>spectrometry.</t> The positions of some proteins associated with the DNMT1 complex are shown. MW: molecular weight markers. b Endogenous DCAF5 and DNMT1 proteins interact with one another. DCAF5 and DNMT1 from HCT116 cells were immunoprecipitated and detected by western blot analyses as indicated. Experiments were repeated three independent times with the same conclusion. c The interaction between DNMT1 and DCAF5 is enhanced by SET7 expression. The SET7 expressing or empty control vectors were transfected into HCT116 cells for 48 h, treated with proteasome inhibitor MG132 (5 μg/ml) for last 6 h, and interactions between DNMT1 and DCAF5 were analyzed by co-immunoprecipitation and western blotting analyses. d Downregulation of DCAF5 prevents DNMT1 degradation triggered by LSD1 deficiency. HCT116 cells were transfected with 50 nM of luciferase siRNA and two independent DCAF5 siRNAs in the presence or absence of LSD1 (LSD1 or L) siRNA. The protein levels of DNMT1, DCAF5, actin, and LSD1 were examined in total <t>cell</t> <t>lysates</t> by western blotting using indicated antibodies and quantified. The P value of LSD1 to control (Luc) siRNAs was calculated by independent Student’s t-test and the P values of double to single knockdowns were calculated by paired Student’s t -test. * P
    Mass Spectrometry Analysis Cell Lysates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mass spectrometry analysis cell lysates/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mass spectrometry analysis cell lysates - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Solution NMR and 31 P and 2 H solid-state NMR analysis. Thin-layer chromatography analysis of Phosphoinositides mix (PIPs). ( A ) REM-CA folds in alpha helix in hydrophobic environment. 2D 1 H- 15 N correlation spectra of REM-CA recorded at 800MHz using the SOFAST-HMQC pulse sequence. All spectra were recorded at 25°C with 1 mM of REMCA-WT in H 2 O/D 2 O (90/10) (red), Dodecylphosphocholine-d38, DPC (blue) and trifluoroethanol-d2, TFE (grey). ( B ) Primulin-stained high-performance thin layer chromatography (HP-TLC) plate of the Phosphoinositides mix (PIPs), reference P6023 SIGMA, along with authentic standards: Phosphatidylserine (PS) Phosphatidylinositol (PI), Phosphatidylinositol-4-phosphate (PI4P) and Phosphatidylinositol-4,5-bisphosphate PI(4,5)P 2 ). ( C ) Histogram representation of relative amounts of each lipid species present in the PIPs quantified by densitometry scanning. Bars indicate SEM, n = 3. ( D ) Temperature effect on REM-CA containing liposomes in the range −8°C to 31°C. Heating curves and corresponding NMR lineshape do not show significant changes. ( E ) 31 P NMR spectra of deuterated 1,2-dimyristoyl-d54- sn -3-phosphocholine (DMPC-d54) membrane in the absence or presence of REM-CA. DOI: http://dx.doi.org/10.7554/eLife.26404.010

    Journal: eLife

    Article Title: Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains

    doi: 10.7554/eLife.26404

    Figure Lengend Snippet: Solution NMR and 31 P and 2 H solid-state NMR analysis. Thin-layer chromatography analysis of Phosphoinositides mix (PIPs). ( A ) REM-CA folds in alpha helix in hydrophobic environment. 2D 1 H- 15 N correlation spectra of REM-CA recorded at 800MHz using the SOFAST-HMQC pulse sequence. All spectra were recorded at 25°C with 1 mM of REMCA-WT in H 2 O/D 2 O (90/10) (red), Dodecylphosphocholine-d38, DPC (blue) and trifluoroethanol-d2, TFE (grey). ( B ) Primulin-stained high-performance thin layer chromatography (HP-TLC) plate of the Phosphoinositides mix (PIPs), reference P6023 SIGMA, along with authentic standards: Phosphatidylserine (PS) Phosphatidylinositol (PI), Phosphatidylinositol-4-phosphate (PI4P) and Phosphatidylinositol-4,5-bisphosphate PI(4,5)P 2 ). ( C ) Histogram representation of relative amounts of each lipid species present in the PIPs quantified by densitometry scanning. Bars indicate SEM, n = 3. ( D ) Temperature effect on REM-CA containing liposomes in the range −8°C to 31°C. Heating curves and corresponding NMR lineshape do not show significant changes. ( E ) 31 P NMR spectra of deuterated 1,2-dimyristoyl-d54- sn -3-phosphocholine (DMPC-d54) membrane in the absence or presence of REM-CA. DOI: http://dx.doi.org/10.7554/eLife.26404.010

    Article Snippet: TLC analysis of phosphoinositides mix (PIPs) Phosphoinositides mix (PIPs), #P6023 SIGMA, was separated by HP-TLC plate along with authentic standards: Phosphatidylserine (PS) Phosphatidylinositol (PI), Phosphatidylinositol-4-phosphate (PI4P) and Phosphatidylinositol-4,5-bisphosphate PI(4,5)P2) ( ).

    Techniques: Nuclear Magnetic Resonance, Thin Layer Chromatography, Sequencing, Staining, High Performance Thin Layer Chromatography

    Time-dependent mass spectrometry analyses of CrGSNOR1 treated with Biotin-maleimide . Recombinant CrGSNOR1 was incubated in the presence of 1 mM Biotin-maleimide. At indicated time points, protein samples were withdrawn and analyzed by MALDI-TOF MS to assess the number of alkylated cysteines. For each alkylated cysteine, the molecular mass of CrGSNOR1 is shifted by +451 Da compared to the native protein (41473.4 Da). Peaks highlighted by an asterisk correspond to the protein-matrix (sinapinic acid) adduct. The y-axis is equal for all mass spectra acquired at times 0, 30, 60, and 90 min, and only indicated in the bottom spectrum.

    Journal: Redox Biology

    Article Title: Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

    doi: 10.1016/j.redox.2020.101806

    Figure Lengend Snippet: Time-dependent mass spectrometry analyses of CrGSNOR1 treated with Biotin-maleimide . Recombinant CrGSNOR1 was incubated in the presence of 1 mM Biotin-maleimide. At indicated time points, protein samples were withdrawn and analyzed by MALDI-TOF MS to assess the number of alkylated cysteines. For each alkylated cysteine, the molecular mass of CrGSNOR1 is shifted by +451 Da compared to the native protein (41473.4 Da). Peaks highlighted by an asterisk correspond to the protein-matrix (sinapinic acid) adduct. The y-axis is equal for all mass spectra acquired at times 0, 30, 60, and 90 min, and only indicated in the bottom spectrum.

    Article Snippet: A five microliters aliquot was kept for MALDI-TOF MS analysis and the rest was used for the enrichment of biotinylated peptides by affinity chromatography.

    Techniques: Mass Spectrometry, Recombinant, Incubation

    Peptide mass fingerprinting of untreated or Biotin-maleimide-treated CrGSNOR1 . Recombinant CrGSNOR1 was incubated in the presence of 1 mM Biotin-maleimide for 20 min and then trypsin digested. The peptide mixture was analyzed by MALDI-TOF MS. Sequence of peptides belonging to CrGSNOR1 is indicated in brackets (numbering according to Fig. 2 ). Cysteines modified by Biotin-maleimide are annotated with the mention “NEM-Bio” and the peak corresponding to the peptide sequence [1−12] of CrGSNOR1 is indicated as N-terminus peptide as it is fused with the 7xHis affinity purification tag.

    Journal: Redox Biology

    Article Title: Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

    doi: 10.1016/j.redox.2020.101806

    Figure Lengend Snippet: Peptide mass fingerprinting of untreated or Biotin-maleimide-treated CrGSNOR1 . Recombinant CrGSNOR1 was incubated in the presence of 1 mM Biotin-maleimide for 20 min and then trypsin digested. The peptide mixture was analyzed by MALDI-TOF MS. Sequence of peptides belonging to CrGSNOR1 is indicated in brackets (numbering according to Fig. 2 ). Cysteines modified by Biotin-maleimide are annotated with the mention “NEM-Bio” and the peak corresponding to the peptide sequence [1−12] of CrGSNOR1 is indicated as N-terminus peptide as it is fused with the 7xHis affinity purification tag.

    Article Snippet: A five microliters aliquot was kept for MALDI-TOF MS analysis and the rest was used for the enrichment of biotinylated peptides by affinity chromatography.

    Techniques: Peptide Mass Fingerprinting, Recombinant, Incubation, Sequencing, Modification, Affinity Purification

    Förster Resonance Energy Transfer assays for peptidoglycan hydrolases. FRET assays were performed with the synthetic peptide Dabcyl-Ala-Glu-Lys-Ala-Gly 5 -EDANS in 20 mM Tris pH 8.0 supplemented with 2 mM CaCl 2 , and measured at 340/490 nm. A) The effect of varying concentration of PlySs2 (0–40 μM) in the presence of 5 μM peptide. B) The effect of varying concentration of peptide (0–10 μM) in the presence of 5 μM PlySs2. C) The kinetics of lysostaphin, ClyS and PlySs2 (5 μM) in the presence of 5 μM peptide. D) The kinetics of ClyS under a different ClyS:peptide ratio. E) The V 0 of PlySs2 at different concentrations of peptide (substrate) was calculated based on the data points from Fig 4B, and plotted in a V 0 /[S 0 ] graph, with a two degree polynomial function used to fit the data points. F) A Lineweaver-Burk plot of 1/V 0 vs 1/[S 0 ], with one outlier removed for clarity. The graph was used for calculating the k cat and K m . All experiments were conducted in triplicates, at least three times, and representative figures are shown.

    Journal: PLoS ONE

    Article Title: Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

    doi: 10.1371/journal.pone.0173919

    Figure Lengend Snippet: Förster Resonance Energy Transfer assays for peptidoglycan hydrolases. FRET assays were performed with the synthetic peptide Dabcyl-Ala-Glu-Lys-Ala-Gly 5 -EDANS in 20 mM Tris pH 8.0 supplemented with 2 mM CaCl 2 , and measured at 340/490 nm. A) The effect of varying concentration of PlySs2 (0–40 μM) in the presence of 5 μM peptide. B) The effect of varying concentration of peptide (0–10 μM) in the presence of 5 μM PlySs2. C) The kinetics of lysostaphin, ClyS and PlySs2 (5 μM) in the presence of 5 μM peptide. D) The kinetics of ClyS under a different ClyS:peptide ratio. E) The V 0 of PlySs2 at different concentrations of peptide (substrate) was calculated based on the data points from Fig 4B, and plotted in a V 0 /[S 0 ] graph, with a two degree polynomial function used to fit the data points. F) A Lineweaver-Burk plot of 1/V 0 vs 1/[S 0 ], with one outlier removed for clarity. The graph was used for calculating the k cat and K m . All experiments were conducted in triplicates, at least three times, and representative figures are shown.

    Article Snippet: Proteins Lysostaphin was purchased from Sigma-Aldrich (St. Louis, MO, USA), ClyS was purified as described elsewhere [ ], and PlySs2 and PlyPy were purified as described in Gilmer et al [ ] and Lood et al [ ], respectively.

    Techniques: Förster Resonance Energy Transfer, Concentration Assay

    Cleavage patterns of different staphylococcal endopeptidases using FRET-labeled and native peptides. Peptidoglycan-like peptides (A) were incubated overnight with buffer only (B, E), lysostaphin (C, F), or PlySs2 (D, G). Both FRET-labeled (B-D), and native peptides (E-G) were used. Generated fragments were detected using LC-MS and analyzed using Skyline. Panels B through G: Measured signals are shown on the y-axis. N-terminal fragments are shown as positive values (black bars) while fragments corresponding to the C-termini are shown with light grey bars. Because the C-terminal signals were found to be very low, the values were multiplied by 50 for visualization purposes. Standard deviations for the replicated measurements are shown. The x-axis shows the origin of the signal with respect to the peptidoglycan-like peptide.

    Journal: PLoS ONE

    Article Title: Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

    doi: 10.1371/journal.pone.0173919

    Figure Lengend Snippet: Cleavage patterns of different staphylococcal endopeptidases using FRET-labeled and native peptides. Peptidoglycan-like peptides (A) were incubated overnight with buffer only (B, E), lysostaphin (C, F), or PlySs2 (D, G). Both FRET-labeled (B-D), and native peptides (E-G) were used. Generated fragments were detected using LC-MS and analyzed using Skyline. Panels B through G: Measured signals are shown on the y-axis. N-terminal fragments are shown as positive values (black bars) while fragments corresponding to the C-termini are shown with light grey bars. Because the C-terminal signals were found to be very low, the values were multiplied by 50 for visualization purposes. Standard deviations for the replicated measurements are shown. The x-axis shows the origin of the signal with respect to the peptidoglycan-like peptide.

    Article Snippet: Proteins Lysostaphin was purchased from Sigma-Aldrich (St. Louis, MO, USA), ClyS was purified as described elsewhere [ ], and PlySs2 and PlyPy were purified as described in Gilmer et al [ ] and Lood et al [ ], respectively.

    Techniques: Labeling, Incubation, Generated, Liquid Chromatography with Mass Spectroscopy

    Addition of FRET-molecules to the synthetic peptides increases hydrophobicity. Native (A, C) and FRET-labeled (B, D) peptides were digested with lysostaphin, and separated by reversed phase C18 chromatography. Samples were loaded onto the column using 0.1% formic acid for 6 minutes, where after the solvent composition was changed to 40% buffer B (acetonitrile, 0.1% formic acid) and 60% buffer A (0.1% formic acid) over 14-minutes, depicted in the figure (see right y-axis for scale). Selected extracted ion trace representing N- and C-terminal fragments for the primary hydrolyzed peptides are shown.

    Journal: PLoS ONE

    Article Title: Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

    doi: 10.1371/journal.pone.0173919

    Figure Lengend Snippet: Addition of FRET-molecules to the synthetic peptides increases hydrophobicity. Native (A, C) and FRET-labeled (B, D) peptides were digested with lysostaphin, and separated by reversed phase C18 chromatography. Samples were loaded onto the column using 0.1% formic acid for 6 minutes, where after the solvent composition was changed to 40% buffer B (acetonitrile, 0.1% formic acid) and 60% buffer A (0.1% formic acid) over 14-minutes, depicted in the figure (see right y-axis for scale). Selected extracted ion trace representing N- and C-terminal fragments for the primary hydrolyzed peptides are shown.

    Article Snippet: Proteins Lysostaphin was purchased from Sigma-Aldrich (St. Louis, MO, USA), ClyS was purified as described elsewhere [ ], and PlySs2 and PlyPy were purified as described in Gilmer et al [ ] and Lood et al [ ], respectively.

    Techniques: Labeling, Chromatography

    Regulation of DNMT1 protein stability by DCAF5. a Isolated DNMT1 protein complexes contain DDB1, DCAF5, and L3MBTL3 proteins (see Supplementary Table 1 for additional information). The Flag-knock-in DNMT1 protein complexes were isolated from HCT116-Flag-DNMT1 cells by anti-Flag M2 antibody affinity resins or from control HCT116 cells without the Flag-DNMT1. The proteins associated with the isolated Flag-DNMT1 complexes were separated in protein gel, excised, trypsinized, and derivative peptides were fractionated by nano-liter liquid chromatography and identified by mass spectrometry. The positions of some proteins associated with the DNMT1 complex are shown. MW: molecular weight markers. b Endogenous DCAF5 and DNMT1 proteins interact with one another. DCAF5 and DNMT1 from HCT116 cells were immunoprecipitated and detected by western blot analyses as indicated. Experiments were repeated three independent times with the same conclusion. c The interaction between DNMT1 and DCAF5 is enhanced by SET7 expression. The SET7 expressing or empty control vectors were transfected into HCT116 cells for 48 h, treated with proteasome inhibitor MG132 (5 μg/ml) for last 6 h, and interactions between DNMT1 and DCAF5 were analyzed by co-immunoprecipitation and western blotting analyses. d Downregulation of DCAF5 prevents DNMT1 degradation triggered by LSD1 deficiency. HCT116 cells were transfected with 50 nM of luciferase siRNA and two independent DCAF5 siRNAs in the presence or absence of LSD1 (LSD1 or L) siRNA. The protein levels of DNMT1, DCAF5, actin, and LSD1 were examined in total cell lysates by western blotting using indicated antibodies and quantified. The P value of LSD1 to control (Luc) siRNAs was calculated by independent Student’s t-test and the P values of double to single knockdowns were calculated by paired Student’s t -test. * P

    Journal: Nature Communications

    Article Title: Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase

    doi: 10.1038/s41467-018-04019-9

    Figure Lengend Snippet: Regulation of DNMT1 protein stability by DCAF5. a Isolated DNMT1 protein complexes contain DDB1, DCAF5, and L3MBTL3 proteins (see Supplementary Table 1 for additional information). The Flag-knock-in DNMT1 protein complexes were isolated from HCT116-Flag-DNMT1 cells by anti-Flag M2 antibody affinity resins or from control HCT116 cells without the Flag-DNMT1. The proteins associated with the isolated Flag-DNMT1 complexes were separated in protein gel, excised, trypsinized, and derivative peptides were fractionated by nano-liter liquid chromatography and identified by mass spectrometry. The positions of some proteins associated with the DNMT1 complex are shown. MW: molecular weight markers. b Endogenous DCAF5 and DNMT1 proteins interact with one another. DCAF5 and DNMT1 from HCT116 cells were immunoprecipitated and detected by western blot analyses as indicated. Experiments were repeated three independent times with the same conclusion. c The interaction between DNMT1 and DCAF5 is enhanced by SET7 expression. The SET7 expressing or empty control vectors were transfected into HCT116 cells for 48 h, treated with proteasome inhibitor MG132 (5 μg/ml) for last 6 h, and interactions between DNMT1 and DCAF5 were analyzed by co-immunoprecipitation and western blotting analyses. d Downregulation of DCAF5 prevents DNMT1 degradation triggered by LSD1 deficiency. HCT116 cells were transfected with 50 nM of luciferase siRNA and two independent DCAF5 siRNAs in the presence or absence of LSD1 (LSD1 or L) siRNA. The protein levels of DNMT1, DCAF5, actin, and LSD1 were examined in total cell lysates by western blotting using indicated antibodies and quantified. The P value of LSD1 to control (Luc) siRNAs was calculated by independent Student’s t-test and the P values of double to single knockdowns were calculated by paired Student’s t -test. * P

    Article Snippet: Immuno-affinity purification and mass spectrometry analysis Cell lysates from twenty dishes (150 mm × 25 mm) of HCT116 cells containing the knock-in Flag-DNMT1 or equal dishes of control HCT116 cells were employed for immuno-affinity purification using 1 ml of the anti-Flag M2 affinity gel (Sigma, A2220) , .

    Techniques: Isolation, Knock-In, Liquid Chromatography, Mass Spectrometry, Molecular Weight, Immunoprecipitation, Western Blot, Expressing, Transfection, Luciferase