Fatty aldehydes were extracted and derivatized according to the previous reported method [32 (link)]. In brief, the pork sample was homogenized in ice-cold methanol containing a proper concentration of DMAQ-13C/15N-aldehydes. The supernatant was collected by centrifugation. Twenty microliters of meat extract and 20 μL of 5 mM DMAQ-12C/14N solution was added into 60 μL of the mixture of methanol and water (1:1, υ:υ) containing 0.1% formic acid. The reaction mixture was incubated at 20 °C for 15 min, and then concentrated under vacuo and reconstructed in 1 mL of acetonitrile. Aldehydes analyses were performed using an ACQUITYTM UPLC equipped with a 6500 QTRAP Triple Quadrupole Mass Spectrometer (SCIEX, Framingham, MA, USA). Chromatograph separation was achieved via ACQUITYTM Premier BEH C18 column (2.1 × 150 mm, 1.7 μm particle size and 130 Å pore size). Mobile phase A and B were deionized water and acetonitrile, respectively. Both contained 0.1% formic acid. Gradient elution (t = 0 min, 25% B; t = 2 min, 28% B; t = 5 min, 60% B; t = 8 min, 100% B: t = 11 min, 100% B; t = 11.1 min, 25% B; t = 12 min, 25% B) was carried out at 300 μL/min. The column temperature was set at 45 °C. The MS parameters were set as follows: IS voltage of 5.5 kV, turbo source gun temperature of 450 °C and CUR of 30 psi.
Qtrap 6500 triple quadrupole mass spectrometer
Manufactured by AB Sciex
Sourced in United States, Germany
The QTRAP 6500+ is a triple quadrupole mass spectrometer designed for quantitative and qualitative analysis. It features high-performance ion optics, fast scanning capabilities, and advanced software for data acquisition and analysis. The QTRAP 6500+ is capable of performing multiple reaction monitoring (MRM) and enhanced product ion (EPI) experiments to provide sensitive and selective detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Dionex ics 6000 hpic system, by Thermo Fisher Scientific (5 mentions)
Prominence uflc hplc system, by Shimadzu (2 mentions)
Multiquant 3, by AB Sciex (2 mentions)
Agilent 1260 series binary hplc system, by Agilent Technologies (2 mentions)
Luna c18 2 column, by Phenomenex (2 mentions)
Analyst software, by AB Sciex (2 mentions)
Multiquant v3, by AB Sciex (1 mentions)
Amide hilic chromatography, by Waters Corporation (1 mentions)
Speedvac, by Eppendorf (1 mentions)
Agilent 1290 uplc system, by Agilent Technologies (1 mentions)
Turboionspray electrospray ionization interface, by AB Sciex (1 mentions)
Capcell core aq column, by Shiseido (1 mentions)
Triversa nanomate, by Advion (1 mentions)
Cardiolipin mix 1, by Avanti Polar Lipids (1 mentions)
7890b gc system, by Agilent Technologies (1 mentions)
Konelab prime60i, by Thermo Fisher Scientific (1 mentions)
Cedex hires analyzer, by Roche (1 mentions)
Matlab, by MathWorks (1 mentions)
Agilent 1290 infinity 2 lc, by Agilent Technologies (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
27 protocols using qtrap 6500 triple quadrupole mass spectrometer
1
Fatty Aldehydes Quantification in Pork
2
LC-MS/MS Quantification of Biomolecules
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A Prominence 20AD liquid chromatography (LC) system (Shimadzu, Columbia, MD) consisting of two LC-20ADXR pumps, a SIL-20 ACXR autosampler, a CTO-20AC controller, and a CBM-20A Lite column oven coupled to a 6500 QTRAP triple quadrupole mass spectrometer (SCIEX, Framingham, MA) was used for LC-MS/MS analysis. For each run, 5 μL was injected onto a C18 XBridge Peptide BEH C18 analytical column (100 mm, 2.1 mm ID, 3.5 μm particle size) connected to a VanGuard XBridge BEH C18 (5 mm, 2.1 mm ID, 3.5 μm particle size) guard column (both Waters, Milford, MA). The column temperature was set at 40 °C. Mobile phase A consisted of 2% (v/v) acetonitrile and 0.1% (v/v) FA in water and mobile phase B consisted of 5% (v/v) water and 0.1% (v/ v) FA in acetonitrile. A constant flow rate of 0.25 mL/min was maintained with a gradient of 2-5% B for the first 5 min with Journal of Proteome Research increments of 10-20% B for 10 min, 20-45% for 4 min, 90% B for 0.5 min, and maintained at 90% B for 3 min before reequilibration at 2% B for 3 min. The total LC time was 26 min. SRM was performed in positive ionization mode at unit resolution for the first and third quadrupoles using a scheduled SRM method with 90 s windows (Supporting Information Table S2). Data acquisition was performed using Analyst software version1.6.2 (Sciex, 2013).
3
Metabolomics Analysis of LncRNA-TBP Overexpression
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LncRNA-TBP overexpression gastrocnemius samples (n = 6) were used for metabolites extraction and then performed on HPIC-MS/MS analysis. The high-performance ion-exchange liquid chromatography (HPIC) separation was carried out using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Fisher Scientific, IL, USA). An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex, USA), equipped with electrospray ionization (ESI) interface, was applied for assay development.
Metabolic hierarchical clustering analysis (HCA) was performed using Cluster3.0 software as previously described [21 (link)].
Metabolic hierarchical clustering analysis (HCA) was performed using Cluster3.0 software as previously described [21 (link)].
4
PPARGC1A Metabolite Profiling in Gastrocnemius
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PPARGC1A under–expression gastrocnemius samples (n = 4) were used for metabolite extraction, and then performed in HPIC-MS/MS analysis. The high-performance ion exchange liquid chromatography (HPIC) separation was carried out using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Fisher Scientific, Waltham, MA, USA). An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex, Framigham, MA, USA) equipped with an electrospray ionization (ESI) interface was applied for assay development. Metabolic hierarchical clustering analysis (HCA) was performed using Cluster 3.0 software.
5
Metabolomics profiling of ZFP36L2-AS knockdown
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ZFP36L2-AS knockdown gastrocnemius samples (n = 6) were used for metabolites extraction, and then performed on HPIC-MS/MS analysis. The high-performance ion exchange liquid chromatography (HPIC) separation was carried out using an Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Fisher Scientific, IL, USA). An AB SCIEX 6500 QTRAP + triple quadrupole mass spectrometer (AB Sciex, USA), equipped with an electrospray ionization (ESI) interface, was applied for assay development.
Metabolic hierarchical clustering analysis (HCA) was performed using Cluster3.0 software as previously described [24 (link)].
Metabolic hierarchical clustering analysis (HCA) was performed using Cluster3.0 software as previously described [24 (link)].
6
Metabolite Extraction and HPIC-MS/MS Analysis
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Tissue samples were added to 500 µL of precooled MeOH/H2O (3/1, v/v). They were then homogenized for 4 min at 40 Hz and sonicated for 10 min in an ice water bath. Homogenization and sonication cycles were repeated three times, followed by incubation at −40 °C for 1 h and centrifugation at 12000 rpm and 4 °C for 15 min. The supernatants (400 µL) were collected and dried. Then, 250 µL of water was added to the dried residue to create a reconstituted solution. The reconstituted samples were vortexed before filtration through a filter membrane, and subsequently transferred to inserts in injection vials for HPIC‐MS/MS analysis. HPIC separation was performed using a Thermo Scientific Dionex ICS‐6000 HPIC System (Thermo Scientific) equipped with Dionex IonPac AS11‐HC (2× 250 mm) and AG11‐HC (2 mm×50 mm) columns. An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex) equipped with an electrospray ionization (ESI) interface was used for assay development. The typical ion source parameters were IonSpray voltage = ‐4500 V, temperature = 450 °C, ion source gas 1 = 45 psi, ion source gas 2 = 45 psi, and curtain gas = 30 psi. AB SCIEX Analyst Work Station software (1.6.3 AB SCIEX), MultiQuant 3.0.3 software, and Chromeleon7 were employed for MRM data acquisition and processing.
7
Metabolomic Analysis of LncEDCH1 Knockdown
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The gastrocnemius samples (n = 7) of LncEDCH1 knockdown were used for metabolite extraction and then subjected to high-performance ion exchange liquid chromatography (HPIC)-tandem mass spectrometry analysis. The HPIC separation was carried out using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Fisher Scientific). An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex, USA), equipped with electrospray ionization interface, was applied for assay development.
Metabolic HCA was performed using Cluster3.0 software as previously described.67 (link)
Metabolic HCA was performed using Cluster3.0 software as previously described.67 (link)
8
Metabolite Profiling of Nutrient Starvation
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Cells were starved of arginine or leucine for the indicated time points as described previously. All conditions received fresh media with fresh dialyzed FBS at each time point. Cells were then washed once with ice-cold PBS on ice, then flash frozen in liquid nitrogen, and placed on dry ice. Cells were stored at −80 °C. 1.5 ml of 80% methanol was added to each 6 cm plate and cells were incubated at −80 °C for 1 h. Plates were scraped for cell harvesting and centrifuged at 4 °C. The supernatant was speed vac dried, and the metabolites were resuspended in 20 ml of LC/MS grade water and 5 ml were injected via targeted LC-MS/MS performed using a 6500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) with Amide HILIC chromatography (Waters). Data were acquired in selected reaction monitoring mode using positive/negative ion polarity switching for steady-state polar profiling of 294 molecules. Peak areas from the total ion current for each metabolite selected reaction monitoring transition were integrated using MultiQuant v3.0 software (AB/SCIEX).
9
Quantitative Metabolomic Analysis of NRK-49F Cells
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Biological triplicate 10 cm2 dishes were used to cultivate NRK-49F cells in full culture medium. Metabolites were extracted using 1 mL of ice-cold 80% methanol on dry ice. Subsequently, the samples were centrifuged at 14,000 rpm for 5 min. To ensure thorough extraction, the cell pellets were subjected to an additional extraction with 0.5 mL of 80% methanol. For accurate protein quantitation, the cell pellets were dissolved in an 8 M urea solution. The supernatant obtained from the metabolite extraction was desiccated into a pellet using SpeedVac from Eppendorf (Hamburg, Germany), using a heat-free technique. Before analysis, the dried pellets were re-suspended in 20 μL of HPLC-grade water in preparation for mass spectrometry, as described before [31 (link)]. A volume of 5–7 μL of the resulting resuspension was injected and subjected to analysis using a cutting-edge hybrid 6500 QTRAP triple quadrupole mass spectrometer from AB/SCIEX (Framingham, MA, USA), which was coupled to a Prominence UFLC HPLC system from Shimadzu (Kyoto, Japan). The analysis was carried out through selected reaction monitoring (SRM), targeting a comprehensive set of 298 endogenous water-soluble metabolites, enabling a thorough examination of the steady-state characteristics of the samples. The data have been deposited to MetaboLights (MTBLS8281) [32 (link)].
10
Quantification of bioactive compounds in plasma
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The plasma concentrations of all components were determined using LC–MS/MS. The instruments consisted of an Agilent 1290 UPLC system (Agilent Technologies, Santa Clara, CA, USA) with a 6500QTRAP triple quadrupole mass spectrometer fitted with a TurboIonSpray electrospray ionization interface (AB Sciex, Framingham, MA, USA). A CAPCELL CORE AQ column (150 × 2.1 mm i.d., 2.7 μm particle size; Shiseido, Tokyo, Japan) was used for all components. The mobile phase consisted of 10 mM ammonium acetate (solution A) and acetonitrile (solution B) at a flow rate of 0.3 mL/min.
For quantification of 6,7-dimethylesculetin, geniposide and genipin, 50 µL of plasma was mixed with 50 µL of water and acetonitrile (4:1, v/v), 10 µL of internal standard solution (100 ng/mL niflumic acid), and 300 µL of acetonitrile. The mixture was centrifuged at 1800×g at 4 °C for 15 min. The supernatant was collected in a test tube at 40 °C in a dry bath under a stream of nitrogen gas. The residue was resolved with 100 μL of water and acetonitrile (9:1, v/v). An aliquot (20 µL) of each sample was injected onto the analytical column.
For quantification of 6,7-dimethylesculetin, geniposide and genipin, 50 µL of plasma was mixed with 50 µL of water and acetonitrile (4:1, v/v), 10 µL of internal standard solution (100 ng/mL niflumic acid), and 300 µL of acetonitrile. The mixture was centrifuged at 1800×g at 4 °C for 15 min. The supernatant was collected in a test tube at 40 °C in a dry bath under a stream of nitrogen gas. The residue was resolved with 100 μL of water and acetonitrile (9:1, v/v). An aliquot (20 µL) of each sample was injected onto the analytical column.
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