We used two different methods of LC–MS analysis for identifying HiPMO1 reaction products. (i) Full Scan LC–MS: We analyzed HiPMO1 reaction products using Full Scan LC–MS with a HPLC analysis system and a LC–MS-2020 mass spectrometer (Shimadzu). We performed HPLC analysis on a C18 column (Agela Odyssil C18, 2.1 × 100 mm) with methanol and water (90:10, v/v), containing 0.2% formic acid as a mobile phase at a flow rate of 0.2 mL/min. Mass spectra were acquired in their positive and negative mode, respectively. The full scan m/z ranged from 100 to 300. (ii) SIM LC–MS/MS: We analyzed HiPMO1 reaction products using SIM LC–MS (ACQUITY UPLC and Q-TOF MS Premier, Waters) on a U3000-HPLC C18 column (Agilent Zorbax SB-C18, 150 × 4.6 mm). The SIM LC–MS settings were as follows: mobile phase A: acetonitrile (5–100%); mobile phase B: water; time: 50 min; flow rate: 0.5 mL/min; ESI mode: positive and negative ionization SIM mode; selected molecular ions: glucuronic acid (m/z 194), saccharic acid (m/z 210), and saccharic acid lactone (m/z 192). Collision-induced dissociation was used for the MS/MS analysis.
Q tof ms premier
Manufactured by Waters Corporation
Sourced in United States
The Q-TOF MS Premier is a high-resolution mass spectrometer designed for accurate mass measurement and identification of unknown compounds. It utilizes quadrupole time-of-flight (Q-TOF) technology to provide precise mass data and structural information.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc system, by Waters Corporation (3 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Lcms 2020 mass spectrometer, by Shimadzu (1 mentions)
Zorbax sb c18, by Agilent Technologies (1 mentions)
Kinetex c18 analytical column, by Phenomenex (1 mentions)
Evo c18 100 a, by Phenomenex (1 mentions)
Avance 400 spectrometer, by Bruker (1 mentions)
Protein lynx global server v2, by Waters Corporation (1 mentions)
996 photodiode array detector, by Waters Corporation (1 mentions)
Bond elut c18 column, by Agilent Technologies (1 mentions)
Nova pak hr c18 column, by Waters Corporation (1 mentions)
6 protocols using q tof ms premier
1
Quantifying HiPMO1 Reaction Products
2
Metabolite profiling of plant leaves
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Freeze-dried leaves (30 mg per sample) were homogenized on ice in 1 ml of MeOH:H2O (10:90) containing 0.01% of HCOOH with metal balls (2 mm ø). The homogenates were centrifuged at 14000 rpm for 20 min at 5 °C. The supernatant was recovered and filtrated through 0.2 µm of regenerated cellulose filters (Teknokroma). An aliquot (20 µl) of the filtered extract was used for LC–MS analysis. Full metabolomics profiling was carried out by non-targeted analysis using an Acquity UPLC system (Waters, Milford, MA, USA) interfaced to a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF MS Premier). The LC separation was performed with a Kinetex C18 analytical column, 1.7 µm particle size, 50 mm × 2.1 mm (Phenomenex). Elution of metabolites was performed using a gradient of methanol and water, both containing 0.01% of HCOOH. The gradient started with an aqueous solvent al 95% and a flow of 0.3 ml min−1. The gradient reached 50% of aqueous solvent at 8 min, increasing the level of organic solvent to 95% at 12 min. The gradient was kept in isocratic conditions for 1 min and later returned to initial conditions in 2 min. The column could equilibrate for 3 min, for a total of 22 min per sample. The library of compounds used for straight compound identification as well as the Q-TOF MS parameters were set as described by Gamir et al. (2014) [49 (link)].
3
Metabolomic Analysis of Plant Samples
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The metabolomic analysis was carried out with six biological replicates per treatment. Fifty milligrams of freeze-dried leaf or root material were extracted at 4 °C with 1 ml of MeOH:H2O (10:90) containing 0.01% of HCOOH. After the centrifugation at full speed at 4 °C for 15 min, the supernatant was filtered through 0.2 μm cellulose filters (Regenerated Cellulose Filter, 0.20 μm, 13 mm D. pk/100; Teknokroma). 20 μl were injected into an Acquity UPLC system (Waters, Mildford, MA, USA) interfaced with a hybrid quadrupole time-of-flight instrument (QTOF MS Premier). Subsequently, a second fragmentation function was introduced into the TOF analyser to identify the signals detected. This function was programmed in a t-wave ranging from 5 to 45 eV to obtain a fragmentation spectrum of each analyte (Gamir et al., 2014) (link). Positive and negative electrospray signals were analysed independently to obtain a global view of the data conduct. To elute analytes, a gradient of methanol and water containing 0.01% HCOOH was used. Three independent biological replicates per treatment, each with three technical replicates, were randomly injected. The LC separation was performed using an UPLC Kinetex 2.6 μm particle size EVO C18 100 A, 50 x 2.1 mm (Phenomenex). Chromatographic conditions and solvent gradients and further were established as described by Gamir et al. (2014) (link).
4
Purification and Characterization of Organic Compounds
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Solvents, reagents and intermediates that were commercially available were purchased and used as received. Dry solvents were purchased in Sure Seal bottles stored over molecular sieves. 1H NMR and 13C spectra were measured on a Bruker Avance 400 spectrometer at 25 °C using DMSO-d6 and CDCl3 as solvents, and chemical shifts are reported as ppm downfield from Me4Si with the number of protons, multiplicities and coupling constants in hertz indicated parenthetically. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), multiplet (m), broadened (b), or a combination thereof. Mass spectroscopy (MS) data were collected using a Waters Q-TOF Premier MS with electron spray ionization. Analytical thin-layer chromatography (TLC) was performed on 0.20 mm silica gel F-254 plates (Qingdao Haiyang Chemical, China). Silica gel 60 with a 300–400 mesh (Qingdao Haiyang Chemical, China) was used and visualization was performed under a UV lamp (254/365 nm). The purity of the final compound was assessed at a wavelength of 210 nm, 254 nm, 268 nm, 230 nm by HPLC analysis. The system was equipped with a DAD UV detector and an Dionex SN: 005581 acclaim @ 120 C18 column (5 μm, 120 Å, 4.6 × 250 mm), and the separations were achieved at rt. The HPLC gradient program utilized 60% MeCN in H2O over 16 min with a 1 mL min−1 flow rate. The purity of target compound was >95% by HPLC.
5
High-resolution UPLC-MS/MS Proteomics
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Reverse-phase ultra performance-liquid chromatography (UPLC), using C-18 columns, was coupled to a Q-TOF Premier™ MS (Waters) system through a nano-electron spray ionization (ESI) online emitter (see Additional file 1 , page 2). LC-MSE data was processed using ProteinLynx Global Server v.2.5 (Waters Corporation) and Rosetta Elucidator v.3.3 (Rosetta Biosoftware, Seattle, WA). Criteria for protein identification were ≥3 fragment ions per peptide, ≥7 fragment ions per protein and ≥2 peptides per protein.
6
Labeling Protocols for PET and SPECT Imaging
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