NPNA was synthesized by the standard Fmoc-solid phase peptide synthesis method and purified by HPLC, using a preparative reverse-phase C18 column (Hypersil Gold 250 × 20 mm, Thermo Scientific, Bremen, Germany). The purity of peptide was greater than 98% and was tested by ESI-MS (Esquire 3000, Bruker Daltonics, Bremen, Germany).
Esquire 3000
Manufactured by Bruker
Sourced in Germany
The Esquire 3000 is a compact, high-performance ion trap mass spectrometer designed for a wide range of analytical applications. It provides stable and sensitive detection of ions within a broad mass range. The Esquire 3000 is characterized by its robust construction, ease of use, and versatile functionality.
Automatically generated - may contain errors
Lab products found in correlation
Jupiter c18 column, by Phenomenex (2 mentions)
Dataanalysis, by Bruker (2 mentions)
Diode array detector, by Agilent Technologies (2 mentions)
1100 hplc system, by Agilent Technologies (2 mentions)
Hypersil gold 250 20 mm, by Thermo Fisher Scientific (1 mentions)
Advance 3 400 mhz, by Bruker (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Human umbilical vein endothelial cells (huvec), by PromoCell (1 mentions)
Avance 3 hd 400, by Bruker (1 mentions)
Hp 1100 series instrument, by Bruker (1 mentions)
Zorbax sil 5 μm column, by Agilent Technologies (1 mentions)
Waters 2424, by Waters Corporation (1 mentions)
Nicolet avatar 360 ft ir spectrometer, by Thermo Fisher Scientific (1 mentions)
Chn 2400, by PerkinElmer (1 mentions)
Amx 300, by Bruker (1 mentions)
Hplc system, by Merck Group (1 mentions)
Agilent 1100 series lc, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Esi l tuning mix, by Agilent Technologies (1 mentions)
Spectrum 3 ftir spectrometer, by PerkinElmer (1 mentions)
49 protocols using esquire 3000
1
Synthesis and Purification of NPNA Peptide
2
Enzyme assay for brain fractions
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Enzyme assays were performed in 0.5-mL Eppendorf tubes. Each homogenized brain fraction was diluted 1:5 with 20 mM Tris–HCl buffer, pH 7.5, and the reaction was initiated by the addition of 5 μg of NPNA and continued by mixing at 37 °C for 120 min (or for 300 min for preparing time course metabolic process). Five-microliter aliquots were taken from the incubation mixture, acidified by 30% MeOH with 0.1% FA and analyzed by ESI-MS (Bruker Esquire 3000) equipped with an autosampler (Famos, Thermo Scientific, earlier LC-Packings, Bremen, Germany). MS/MS analysis was performed to confirm the sequence of the released fragments. Quantitation of the enzyme activity was performed by integration of the peak area of formed products on the mass chromatogram. The samples were incubated with and without amastatin, an aminopeptidase inhibitor.
3
Tandem MS Analysis of Ag+ Complexes
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Tandem MS experiments were carried out in Rome using a Paul ion trap (Esquire 3000+, Bruker Daltonics, Bremen, Germany) coupled to an ESI source working in positive polarity mode in the m/z 50–500 range. For each analysis, 40 scans were coadded with an accumulation time of 5 ms. In particular, peaks at m/z 214 and 321 (corresponding to the monoisotopic Ag+(BA) and Ag+(BA)2 clusters, respectively) were mass isolated and fragmented by varying the resonance excitation amplitude between 0.05 and 0.70 V, to evaluate the appearance of competitive and/or consecutive fragmentation pathways.[56] Helium was used as collision gas at a nominal pressure of 1.5×10−6 mbar.
4
APCI Mass Spectrometry of Chloroform Samples
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The positive-ion APCI mass spectra were measured on an ion trap analyzer Esquire 3000 (Bruker Daltonics, Bremen, Germany) within the mass range m/z = 50-1000. Samples were dissolved in chloroform and analyzed by direct infusion at the flow rate of 40 mL/min. The ion source temperature was 300 °C, the APCI probe temperature was 350 °C, the flow rate and the pressure of nitrogen were 3 L/min and 25 psi, respectively. For MS/MS measurements, the collision amplitude was 0.9 V and the isolation width of precursor ions was 4 m/z.
5
Analytical Characterization of Organic Compounds
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Reaction progress was monitored using analytical thin-layer chromatography (TLC) on Merck silica gel 60 F-254 plate. Visualization was achieved by UV light (254 nm). Flash chromatography was performed with Scharlau silica gel 60 (0.04–0.06 mm) packing. 1H NMR and 13C NMR spectra were recorded on a Bruker Advance III 400 MHz instrument. Signals are quoted as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), and m (multiplet). Chemical shifts (δ) are expressed in parts per million, relative to solvent resonance as the internal standard. Coupling constants (J) are in hertz (Hz). Original 1H NMR and 13C NMR spectra for final compounds are shown in Supplementary Materials (Figure S2 ) Melting points were determined on a Stuart Scientific (BIBBY) melting point apparatus. Final products were analyzed for purity by reverse-phase high-performance liquid chromatography (HPLC) on Agilent Technologies 1260 Infinity II apparatus (Figure S10 ). Mass spectrometry was performed by the CEMBIO Analytical Service Laboratory of the Universidad CEU San Pablo on an MS/IT Esquire 3000 Bruker Daltonics apparatus. Mass spectra reports for final compounds are shown in Supplementary Materials (Figure S11) .
6
Mass Spectrometric Characterization of Conjugates
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Mass spectrometric experiments were performed by electrospray ionization on a Bruker Daltonics Esquire 3000+ (Bruker Daltonic GmbH, Bremen, Germany) ion trap mass spectrometer, operating with continuous sample injection at 10 μL/min flow rate. Mass spectra were recorded in positive ion mode in the m/z 50–2000 range, with 250 °C heating and N2 as nebulizer (10 psi) and dry gas (4 L/min). Stock solutions were prepared in double-distilled water (cstock = 1 nmol/µL) and diluted with the solvent mixtures. Samples were analyzed in acetonitrile-water (50:50%, v/v) mixture with or without 0.1% acetic acid (cconj.;acidic = 10 pmol/µL and cconj.;non-acidic = 20 pmol/µL) and solutions containing NH4HCO3 (pH 7.8) or NH4OAc buffers (pH 6.7) and acetonitrile (50:50%, v/v, final buffer concentration was 50 mM; cconj.;buffer = 50 pmol/µL).
7
Synthesis and Characterization of PAMAM Dendrimers
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Materials PAMAM G2 and PAMAM G4 dendrimer 2 (ethylenediamine core, 16 and 64 surface groups respectively, products number: 412406 and 412,449 respectively) and all other organic reactants, solvents and culture reagents were purchased from Sigma-Aldrich (Milan, Italy) if not differently specified and used as received. 2-(Trifluoromethyl)acryloyl chloride 8 was synthesized from 2-(trifluoromethyl)acrylic acid as described in literature [45] (link). Spectra/Por dialysis bags (MWCO = 1 kDa) were from Spectrum laboratories (Compton, CA, USA). Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany). 1 H, 13 C, and 19 F NMR spectra were recorded on 400 MHz spectrometers. Chemical shifts are expressed in ppm (δ), using tetramethylsilane (TMS) as internal standard for 1 H and 13 C nuclei (δ H and δ C = 0.00) while C 6 F 6 was used as external standard (δ F -162.90) for 19 F. ESI mass spectra were performed by a Bruker Esquire 3000 + instrument equipped with a MS detector composed by a ESI ionization source and a Single Quadrupole mass selective detector. Purifications of the intermediates was performed by Flash Chromatography (FC) with Biotage Isolera One Flash Purification Chromatography ISO-1SV Unit4 Pred Selekt.
8
Analytical Characterization of Chemical Products
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Reagents, materials and solvents were obtained from Alfa Aesar, Sigma-Aldrich, Merck, Reanal, or VWR. For moisture-sensitive reactions, the solvents were distilled with the standard procedures or dried on molecular sieves (3 Ǻ). Products were analyzed by reverse-phase HPLC on a Phenomenex Jupiter C-18 column using the water/acetonitrile mixtures of 0.1% TFA in water (A) and 0.08% TFA, and 95% acetonitrile in water (B), and UV detection completed at 220 and 280 nm. Products were identified with Bruker Esquire 3000+ tandem quadrupole mass spectrometer equipped with an electrospray ion source.
9
Characterization of Organometallic Compounds
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NMR spectra were recorded at room temperature (25 °C) with a Bruker AVANCE III HD 400 spectrometer. 1H (400.13 MHz) and 13C (100.16 MHz) NMR spectra were referred to SiMe4 (TMS) as an internal standard. 11B NMR (128.38 MHz) spectra were referenced to the unified Ξ scale [57 (link)]. ESI mass spectra were recorded with a Bruker ESQUIRE 3000 (Benchtop LC Ion trap) mass spectrometer. The FT-IR spectra were obtained with a Nicolette IS5 (ATR) from Thermo Fisher (Waltham, MA, USA) with the scan range 4000–400 cm−1. A Hereaus VARIO EL oven was used to perform elemental analyses.
10
Hyperoxidation Assay of PRX-IIE
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Hyperoxidation of PRX-IIE was assayed as described above using the FOX assay with 400 µM H2O2 as substrate and increasing CuOOH concentrations. Furthermore, the oxidation state was investigated by electrospray ionization coupled with mass spectrometry (ESI-MS; Esquire 3000, Bruker Daltonics, Bremen, Germany). 10-20 µM of prereduced protein in 100 mM Tris-HCl, pH 8.0, was incubated with 5 mM DTT and different CuOOH and 0.5 mM DTT and increasing H2O2 concentrations for 1 h at room temperature (RT). Excess low molecular weight reagents were removed by acetone precipitation and proteins were resuspended in H2O. Dilutions were prepared in 30 % EtOH, 0.1 % formaldehyde (FA) and the mixture was introduced into the ESI-MS.
Instrumental settings: Capillary voltage = 4.000 V. Nebulizer gas pressure = 15 psi. Drying gas flow = 4.0 L/min. Drying gas temperature = 300 °C. Mass-to-charge (m/z) values: 650-1200. Mass spectra were deconvoluted using the software provided by the manufacturer (DataAnalysis, Bruker Daltonics, Bremen, Germany).
Instrumental settings: Capillary voltage = 4.000 V. Nebulizer gas pressure = 15 psi. Drying gas flow = 4.0 L/min. Drying gas temperature = 300 °C. Mass-to-charge (m/z) values: 650-1200. Mass spectra were deconvoluted using the software provided by the manufacturer (DataAnalysis, Bruker Daltonics, Bremen, Germany).
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