The purified hydrolysed lipids were analysed by mass spectrometry using a Q-Trap 2000 (Applied Biosystems, Foster City, USA) which operated at ion-spray ionisation (ESI-MS). The purified lipid hGDNT was diluted to 0.1 μg/mL prior to measurement. IR spectrum of hGDNT was recorded by an ALPHA FT-IR spectrometer (Bruker Corp., Massachusetts, USA). 5 mg of hGDNT was used to obtain the transmission spectrum. For 1H-NMR studies, hydrolysed lipids were dissolved at 2.5 mg/mL in CDCl3 and analysed with a JEOL ECX-400 with an autotune sample head. HPTLC plates were used to identify the lipids PLFE and hGDNT using the mobile phases CHCl3 : MeOH : H2O (22.5 : 50 : 22.5 v/v) and CHCl3 : MeOH (90 : 10 v/v), respectively. Lipids were spotted using a MeOH/sulfuric acid spray reagent.
Q trap 2000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q-Trap 2000 is a high-performance liquid chromatography (HPLC) system designed for quantitative and qualitative analysis. It features a quadrupole ion trap mass spectrometer for accurate detection and identification of analytes. The system is capable of performing multiple reaction monitoring (MRM) and enhanced product ion (EPI) scans to provide reliable and sensitive analytical results.
Automatically generated - may contain errors
Lab products found in correlation
Ecx 400, by JEOL (1 mentions)
Alpha ftir spectrometer, by Bruker (1 mentions)
Voyager de pro, by Thermo Fisher Scientific (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Reverse phase hplc, by Shimadzu (1 mentions)
Aviii 700 mhz instrument, by Bruker (1 mentions)
Turboionspray, by Thermo Fisher Scientific (1 mentions)
1100 series, by Agilent Technologies (1 mentions)
Mercury yh 400 nmr spectrometer, by Agilent Technologies (1 mentions)
Pb 10 digital ph meter, by Sartorius (1 mentions)
6 protocols using q trap 2000
1
Analysis of Purified Hydrolysed Lipids
2
Characterization of Crotalus viridis viridis Venom Proteins
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Lyophilized Cvv venom (10 mg) was dissolved in 1 ml of 20 mM Tris–HCl, 150 mM NaCl, pH 8.8 and centrifuged at 5,000 g for 2 min. The supernatant was applied onto a reverse phase analytical C8 column (5 µm, 250×4.6 mm) (Kromasil, Sweeden), previously equilibrated with the same buffer. Venom proteins were separated by reverse phase HPLC (Shimadzu, Japan). Fractions (0.7 ml/tube) were collected at a 1 ml/h flowrate. A linear gradient of water/acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used. The elution profile was monitored by absorption at 280 nm, and the molecular homogeneity of the relevant fractions was verified by SDS-PAGE. Fractions containing protein peaks were dried in a Speed-Vac (Savant, Thermo Scientific, USA) and resuspended in distilled water prior to protein quantification by the Bradford method. Molecular mass determination was performed by MALDI-TOF and by electrospray ionization (ESI) mass spectrometry using a Voyager-DE Pro and a QTrap 2000 (both from Applied Biosystems), respectively.
3
Enzymatic Acetylation of Apramycin
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ApmA-catalyzed acetylated apramycin was produced from 50-ml in vitro reactions (50 mM HEPES, pH 7.5) consisting of 500 μM aminoglycoside, 500 μM acetyl-CoA, and 1 μM ApmA. Reaction mixtures were incubated at room temperature until acetylated products (mass increase of 42.0 Da) were detected by liquid chromatography (LC)/ESI-MS. Enzymes were removed by centrifugation using an Amicon Ultra-15 centrifugal filter and the flowthrough subsequently concentrated. Acetyl-apramycin was purified from the concentrate using AG50W-X8 strong cation resin. The resin was preequilibrated with 1% NH4OH and washed with H2O until a neutral pH was obtained. Fractions containing acetylated products were identified by LC/ESI-MS, followed by detailed analysis with NMR and HR-ESI-MS. LC/ESI-MS data were acquired using a QTrap 2000 (Applied Biosystems) system equipped with an Agilent 1100 LC interface. HR-ESI-MS data were acquired using an Agilent 1290 ultraperformance liquid chromatography (UPLC) separation module and quadrupole time of flight (Q-TOF) G6550A mass detector in positive ion mode. NMR analysis was completed using a Bruker AVIII 700 MHz instrument in deuterated water as the solvent. The chemical shifts are reported in parts per million.
4
Mass Spectrometry Analytical Protocols
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Either direct mass analysis or analysis through coupling with liquid chromatography was performed with the mass spectrometers, low resolution (Q TRAP 2000, Applied Biosystems, Foster City, CA, USA), and high resolution (Q TOF 1er, Waters, Milford, MA, USA) that were operated in the positive and/or negative mode.
5
Glycopeptide Analysis by HPLC-ESI-MS/MS
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The culture broth of the mutant CK1 was investigated by HPLC-ESI-MS(/MS). For sample preparation an adsorption chromatography with AMBERLITE® XAD16 material was performed. The used HPLC-ESI-MS-MS system consisted of a capillary-LC-system (1100 series, Agilent Technologies Deutschland GmbH, Böblingen, Germany) coupled to a QTrap2000 with a TurboIonSpray source (Applied Biosystems, Darmstadt, Germany). Separations were performed on a Jupiter 4 μm Proteo 90A column system (main column: 150 × 1 mm; precolumn: 30 × 1 mm; Phenomenex, Aschaffenburg, Germany) with a flow rate of 50 μl min–1 in micro mode and the following gradient: t = 0 min: 5% B; t = 10 min: 20% B; t = 13 min: 50% B; t = 14 min: 100% B (solvent A: 0.1% HCOOH in water, solvent B: 0.1% HCOOH in MeCN). The injection volume was 5 μl. The TurboIonSpray source dependent parameters were optimised for the used flow rate of 50 μl min–1 to: CUR 30, IS 5500, nebuliser gas 70, turbo gas 70, TEM 300. The compound dependent parameters were optimised with different glycopeptides to: DP 30, EP 12, CE 10, Q3 entry barrier 12. The EMS scans were carried out in positive mode, with a LIT scan rate of 1000 amu s–1 and dynamic fill time. The EPI scans had the following parameters: Q1 resolution unit, LIT scan rate 1000 amu s–1, fixed LIT fill time 500 ms, CE 30, CES 20, CAD gas high.
6
Characterization of Compounds by Analytical Techniques
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The nuclear magnetic resonance spectroscopy (NMR) of compounds were identified by 1 H NMR and 13 C NMR (Varian Mercury YH-400 NMR spectrometer), using tetramethylsilane (TMS) as an internal standard. ESI mass spectra were obtained using a Q-Trap 2000 (Applied Biosystems Corporation, USA) without using the liquid phase part. MOLDI-TOF mass spectra were obtained using a Autoflex speed TOF by Brucker. The average molecular weight and the molecular weight distribution was determined by GPC (Agilent 1260). All fluorescence measurements were carried out in a 1-cm path length quartz cuvette with a Hitachi F-2700 spectrometer (Shimadzu Corporation, Japan). Measurements of fluorescence quantum yields were carried out with an FLS920 steady state and transient state fluorescence spectrometer (Edinburgh Instrument). All pH measurements were tested with a Sartorius PB-10 digital pH meter. All the optical measurements were carried out at room temperature (298 K) under ambient conditions.
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