dNTP and rNTP concentration (pmol/million cells) for activated CD4+T cells were collected from a previous study having 4 replicates, and the precalculated average was used to represent dNTP/rNTP ratios (41 (link)). The nucleotide samples were extracted based on the established protocol (42 (link)) with some modifications. To prepare the nucleotide samples, 2 × 106 cells of HEK293T or hESC-h9 were counted and centrifugated to obtain a cell pellet. The pellet was washed with PBS and then vortexing was performed for 2 min with 200 μl of cold 65% methanol for cell lysis. The cell mixture was incubated at 95°C for 3 min and then incubated on ice for 1 min to complete the cell lysis. By centrifugation at 14 000 rpm for 3 min, the supernatant containing nucleotides was isolated. To quantify the intracellular dNTPs and rNTPs, an ion pair chromatography-tandem mass spectrometry method (43 (link)) was applied, with modifications. Chromatographic separation and detection were performed on a Vanquish Flex system (Thermo Fisher Scientific) coupled with a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific). Analytes were separated using a Kinetex EVO-C18 column (100 × 2.1 mm, 2.6 μm) (Phenomenex) at a flow rate of 250 μl/min. Pmol/million cells were calculated for rNTPs and dNTPs for all four replicates to calculate the ratios.
Tsq quantiva triple quadrupole mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TSQ Quantiva triple quadrupole mass spectrometer is a high-performance analytical instrument designed for quantitative analysis. It utilizes triple quadrupole technology to provide accurate and sensitive measurement of target analytes in complex samples.
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Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (3 mentions)
Vanquish ultra, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 xrs uhplc system, by Thermo Fisher Scientific (2 mentions)
Tracefinder 3, by Thermo Fisher Scientific (2 mentions)
Microbca, by Thermo Fisher Scientific (2 mentions)
Xcalibur, by Thermo Fisher Scientific (2 mentions)
Vanquish flex system, by Thermo Fisher Scientific (1 mentions)
Kinetex evo c18 column, by Phenomenex (1 mentions)
Masslynx, by Waters Corporation (1 mentions)
Waters acquity uplc, by Waters Corporation (1 mentions)
Quattro micro api tandem mass spectrometer, by Waters Corporation (1 mentions)
Nexera uhplc system, by Shimadzu (1 mentions)
Vanquish uhplc system, by Thermo Fisher Scientific (1 mentions)
Accucore vanquish c18 column, by Thermo Fisher Scientific (1 mentions)
Cortecs c18 column, by Waters Corporation (1 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
0.22 mm filter, by Merck Group (1 mentions)
Security guard cartridge, by Phenomenex (1 mentions)
Qtrap 5500 ms, by AB Sciex (1 mentions)
27 protocols using tsq quantiva triple quadrupole mass spectrometer
1
Quantifying Cellular Nucleotide Levels
2
Liver Metabolite Extraction and LC-MS Analysis
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Liver metabolites were extracted from tissue snap-frozen after dissection using a reported methanol/chloroform method [24 (link)]. Aqueous metabolites were further extracted as described in [24 (link)] and analysed using a Vanquish ultra-high performance liquid chromatography (UHPLC) system and TSQ Quantiva triple quadrupole mass spectrometer (Thermo Scientific) with compounds directly infused. Parameter optimisation used 1 μM standard solutions in a chromatographic buffer. Optimal mass spectrometry parameters and mass transitions were generated by automatic MassLynx™ (Version 1.4, Waters) protocols or, if standards were unavailable, deduced from known analogue parameters.
3
UHPLC-MS/MS Quantitative Analysis
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Thermo Scientific TSQ Quantiva Triple Quadrupole mass spectrometer was interfaced with the Thermo Scientific UltiMate 3000 XRS UHPLC system using a pneumatic-assisted heated electrospray ion source. Data acquisition and analysis were performed using Xcalibur 4.0.
4
Multianalyte Profiling of Lignocellulose Residues
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Total soluble carbohydrates (sum of monomers and oligomers) in the LCR were quantified by GC–MS following acid hydrolysis and alditol acetate derivatization [62 ]. Oligomeric carbohydrates in a separate set of LCR samples were quantified using the NREL Klason lignin protocol [63 ]. Samples were submitted to the Wisconsin State Laboratory of Hygiene for metals analysis. Briefly, LCR samples were digested using a mixture of nitric, hydrochloric, and hydrofluoric acids and hydrogen peroxide. Samples were then diluted and analyzed using a Thermo-Finnigan Element XR™ ICP-MS (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Vitamin content was quantified by reverse-phase LC–MS/MS using a Waters Acquity UPLC with Quattro Micro™ API tandem mass spectrometer (Waters Corporation, Milford, MA, USA). Quantification of amino acids in LCR is described elsewhere [29 ] [64 (link)]. LDIs in the LCR were identified and quantified using LC–MS/MS. Samples were separated using an Acquity UPLC HSS T3 reversed phase column (150 × 2.1 mm, Waters Corp.) at 35 °C with a binary mobile phase (Phase A 0.1% acetic acid, Phase B acetonitrile) and 0.4 mL min−1 flow rate. The liquid chromatography system was connected to a TSQ Quantiva Triple Quadrupole mass spectrometer (Thermo Fisher Scientific, Inc.).
5
Quantifying Glycolytic Flux via LC-MS
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The glycolytic flux was measured based on the rate of glucose consumption and the ratio of 13C incorporated into lactate determined by LC-MS. Briefly, cells were cultured in medium with or without [U-13C6] glucose. After 12 h, medium was collected and cells were treated with cold 80% methanol. Metabolites were extracted and analyzed by LC-MS. Flux analysis was performed on a TSQ Quantiva Triple Quadrupole mass spectrometer (Thermo Fisher) with positive/negative ion switching. MRM mode was used for data acquisition. Mobile phase A was prepared by adding 2.376 mL tributylamine and 0.858 mL acetic acid to HPLC-grade water, then added HPLC-grade water to give a total volume of 1 liter. Mobile phase B was HPLC-grade methanol. Polar metabolites were separated on a Synergi Hydro-RP 100A column with the column temperature at 35 °C. The measured mass isotopomer distributions were corrected according to their natural abundances.
6
Targeted and Untargeted Metabolomics Analysis
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Metabolomics analysis was performed as previously described [24 (link)]. Briefly, the cells were washed twice with cold PBS and incubated with pre-chilled 80% methanol (−80 °C) for 1 h at −80 °C. Then, the cells were scraped in 80% methanol on dry ice and centrifuged for 5 min. The extracted metabolites in the supernatant were dried using a lyophilizer and the protein concentrations of pellets were measured for normalization. The dried metabolites were dissolved in 80% methanol and analyzed by LC–MS/MS. We used the TSQ Quantiva Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with positive/negative ion switching for the quantitative analysis of targeted metabolites. The Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was chosen for untargeted metabolites profiling. Metabolites were identified based on accurate ion masses and MS/MS fragments. Relative quantitation of metabolites was analyzed with TraceFinder 3.2 (Thermo Fisher Scientific, Waltham, MA, USA).
7
Renal Metabolome Profiling via LC-MS/MS
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A total of 20 mg of renal tissue was homogenized in pre-chilled 80% methanol five times using a tissue grinder at 4 °C, with each run lasting 15 s at a frequency of 60 Hz, followed by a 10 s pause. Subsequently, the homogenates were centrifuged to collect the supernatants. Firstly, the metabolites were wholly dried with a lyophilizer. Then, dissolved metabolites were analyzed using LC-MS/MS. The profiling of targeted and untargeted metabolites was conducted using a TSQ Quantiva™ Triple Quadrupole Mass Spectrometer and a Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Metabolites were identified based on retention time and quantitated using Trace-Finder 3.2 (Thermo Fisher Scientific, Waltham, MA, USA). For untargeted profiling, metabolites were identified based on MS/MS matching with the standard library. Two levels of identification were achieved in the analysis, one of which was through MS/MS confirmation and the other via potential assignment according to precursor ion masses. Missing values were imputed using the mean imputation method. The MS metabolomics data are available at www.ebi.ac.uk/metabolights/MTBLS8350 [23 (link)]. All metabolites identified and the relative abundance values are listed in Table S3 .
8
UHPLC-MS/MS Quantification of Compounds
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Samples were prepared by adding 180 µL acetonitrile (containing 1 nM loperamide as internal standard) to 20 µL mouse plasma or fecal extract. Samples were vortexed for 5 min at room temperature and centrifuged at 16,000× g for 10 min at 4 °C. After transfer of the supernatant to a glass vial, 8µL was injected into a Thermo Hypersil C18 column (100 × 2.1 mm, 1.9 µm) on a Shimadzu Nexera UHPLC system, and the separation of compounds was performed using gradient elution from 25% to 95% acetonitrile (with 0.1% formic acid) in 5 min at a flow rate of 0.2 mL/min and oven temperature of 50 °C. The effluent was ionized using positive ion electrospray on a Thermo TSQ Quantiva triple-quadrupole mass spectrometer. The integrated peak areas for these transitions were calculated for each sample using Skyline software (version 21.0, University of Washington). The concentration of compounds in the samples was determined using a linear equation derived from running a set of 7 standards. The limit of detection was approximately 1 nM for each compound.
9
Targeted and Untargeted Metabolomics Analysis
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Metabolomics analysis was performed as previously described [70 (link)]. Briefly, the cells were washed twice with cold PBS and incubated with pre-chilled 80% methanol (−80 °C) for 1 h at −80 °C. Then, the cells were scraped in 80% methanol on dry ice and centrifuged for 5 min. The extracted metabolites in the supernatant were concentrated completely to dryness with a lyophilizer and the protein concentrations of centrifuged pellets were used for normalization. The dried metabolites were dissolved in 80% methanol and analyzed by LC–MS/MS. We used the TSQ Quantiva™ Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with positive/negative ion switching for targeted metabolites quantitative analysis. The Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was chosen for untargeted metabolites profiling. Metabolites were identified based on the retention time and the accurate mass measured with <5 ppm mass accuracy. Quantitative information of metabolites was analyzed by TraceFinder.
10
Pharmacokinetics of CAD-31 in Rats and Mice
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Sprague-Dawley rats had free access to food and water. CAD-31 was given by gavage to rats at 20 mg/kg in corn oil and intravenously in 15% HS 15/PBS. Whole-blood samples were collected from the jugular vein at every time point and brain collected after 20-ml saline perfusion. CAD-31 was extracted with acetonitrile and compared with standards on a TSQ Quantiva™ Triple Quadrupole Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). For the mouse feeding studies, mice had free access to food containing 200 ppm CAD-31. Blood was collected by heart puncture, and the brain concentration was determined as with rats.
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