The metabolites were extracted from MDA-MB-231 cells transfected with Adv-GFP or Adv-hLASS2-GFP via the assistance of MetWare (Wuhan, China). In brief, hydrophilic and hydrophobic compounds were separately extracted and analysed using an LC–ESI–MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/ ; MS, QTRAP® System, https://sciex.com/ ) in accordance with previously published methods [19 (link)]. The mass spectrometry data were processed by Software Analyst 1.6.3. The highly enriched metabolic pathways associated with the differentially abundant metabolites were identified using KEGG and metabolite set enrichment analysis (MSEA).
Qtrap system
Manufactured by AB Sciex
Sourced in United States
The QTRAP® System is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform designed for quantitative and qualitative analysis. It combines a triple quadrupole mass spectrometer with a linear ion trap, providing enhanced sensitivity and selectivity for complex sample analysis.
Automatically generated - may contain errors
Lab products found in correlation
Exionlc ad, by AB Sciex (10 mentions)
Analyst 1, by AB Sciex (7 mentions)
Acquity uplc hss t3 c18, by Waters Corporation (6 mentions)
Qtrap lc ms ms system, by AB Sciex (4 mentions)
Shim pack uflc cbm a system, by Shimadzu (4 mentions)
Esi turbo ion spray interface, by AB Sciex (1 mentions)
Accucore c30, by Thermo Fisher Scientific (1 mentions)
Cbm30a system, by Shimadzu (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Cbm a system, by Shimadzu (1 mentions)
Shim pack uflc, by Shimadzu (1 mentions)
21 protocols using qtrap system
1
Metabolic Profiling of hLASS2 in TNBC
2
LC-ESI-MS/MS Metabolite Profiling
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The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM A system, https://www.shimadzu.com/ ; MS, QTRAP® System, https://sciex.com/ ). The analytical conditions were as follows, UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm * 100 mm); column temperature, 40°C; flow rate, 0.4 ml/min; injection volume, 2 μl; solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 95:5 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 14.0 min. LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: source temperature 500°C; ion spray voltage (IS) 5,500 V (positive), −4,500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) was set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.
3
UPLC-MS/MS Analysis of Compound Extracts
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A LC-ESI-MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/ ; MS, QTRAP® System, https://sciex.com/ ) was used to analyze the sample extracts. Here are the conditions for the analysis, UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm*100 mm); column temperature, 40 °C; flow rate, 0.4 mL/min; injection volume, 2 µL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid); gradient program, 95:5 V/V at 0 min, 10:90 V/V at 10.0 min, 10:90 V/V at 11.0 min, 95:5 V/V at 11.1 min, 95:5 V/V at 14.0 min.
4
LC-MS/MS Analysis of Serum Metabolites
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The serum sample extracts were analyzed using an LC–ESI–MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/ ; MS, QTRAP® System, https://sciex.com/ ). LIT and triple quadrupole (QQQ) scans were acquired with a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® LC–MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: source temperature 500 °C; ion spray voltage (IS) 5500 V (positive), -4500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) set at 55, 60, and 25.0 psi, respectively; and high collision gas (CAD). Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period.
5
Metabolomics Analysis of Muscle Tissue
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Tissue samples of muscle are extracted by Metware according to standard procedures. The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/ ; MS, QTRAP® System, https://sciex.com/ ). LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period. Significantly regulated metabolites between groups were determined by variable importance in projection (VIP)≥1 and absolute Log2FC (fold change)≥1. VIP values were extracted from OPLS-DA result, which also contain score plots and permutation plots, was generated using R package MetaboAnalystR. The data was log transform (log2) and mean centering before OPLS-DA. In order to avoid overfitting, a permutation test (200 permutations) was performed.
6
Metabolomic Profiling of Sheep Colonic Contents
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Sheep’s colonic contents were homogenized using a 500 mL solution of cold methanol and water (70%, vol/vol). Following a 10 min ultrasonic treatment in an ice-water bath, samples were swirled for 3 min and centrifuged for 10 min at 4°C (16,099 × g). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (UPLC, Shim-pack UFLC SHIMADZU CBM A system, https://www.shimadzu.com/ ; MS, QTRAP system, https://sciex.com/ ) was then performed using the obtained supernatants (9 (link)).
7
Metabolomic Analysis of Cd-Treated Gill Tissues
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The gill tissues from the Cd-treated group (7 d) were collected for metabolomic analysis considering that the accumulation of metabolites requires some time. Metabolite extraction and detection were performed in accordance with previously described methods [21 (link)]. Briefly, samples were ground to powder with liquid nitrogen using a mixer mill (MM 400, Retsch, Laichi, Germany). Then, 20 mg of powder for each sample was extracted with 400 μL of aqueous methanol (Methanol/Water = 7:3, v/v) containing internal standard and shaken at 2500 rpm for 5 min. After placing on ice for 15 min, the extract was centrifuged at 12,000× g for 10 min (4 °C). Supernatant (300 μL) was collected and placed in −20 °C for 30 min. The supernatant was collected and transferred to vials for UPLC-QTOF-MS analysis (UPLC, ExionLC AD, https://sciex.com.cn/ (accessed on 21 February 2023); MS, QTRAP® System, https://sciex.com/ (accessed on 21 February 2023)). Significantly changed metabolites (SCMs) were identified with a threshold of VIP values (variable importance in project, VIP > 1) and p-values (p-value < 0.05, Student’s t test). These metabolites were annotated using the KEGG Compound database and mapped to the KEGG Pathway database for pathway analysis.
8
LC-ESI-MS/MS Analytical Procedure for Compounds
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The sample extracts were analyzed using an LC-ESI-MS/MS system (ExionLC AD equipped with a QTRAP System; Sciex, Framingham, MA, USA). The analytical conditions were as follows: column, Thermo Accucore C30 (2.6 μm, 2.1 mm × 100 mm; solvent system A: acetonitrile/water [60/40 (v/v), 0.1% formic acid, 10 mmol/L ammonium formate], and B: acetonitrile/isopropanol [10/90 (v/v), 0.1% formic acid, 10 mmol/L ammonium formate]); gradient program A/B (v/v), 80:20 at 0 min, 70:30 at 2 min, 40:60 at 4 min, 15:85 at 9 min, 10:90 at 14 min, 5:95 at 15.5 min, 5:95 at 17.3 min, 80:20 at 17.3 min, and 80:20 at 20 min; flow rate, 0.35 mL/min; temperature, 45°C; and injection volume, 2 μL. The effluent was alternatively connected to an ESI-triple quadrupole linear ion trap (QTRAP)-MS.
9
Metabolite Profiling of Liver Samples
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The 50-milligram liver sample was placed into the 2-mL EP tube and 500 μL of pre-cooled extractant was added (70% methanol aqueous solution). After homogenizing, the sample was centrifuged at 1500 rpm for 5 min and then allowed to stand for 15 min on ice before being centrifuged again for 10 min at 12000 rpm. Subsequently, 200 μL of the supernatant was collected for analysis using an LC-ESI-MS/MS system (UPLC, Shim-peck UFLC SHIMADZU CBM30A system; MS, QTRAP® System) which was equipped with electrospray ionization (ESI) Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). A specific set of multiple reaction monitoring (MRM) transitions was monitored for each period according to the metabolites eluted within this period.
All data were processed and normalized using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/ ). The samples were grouped using principal component analysis (PCA) and significantly up-regulated and down-regulated metabolites between groups were determined by variable importance in the projection (VIP) ≥ 1 and absolute Log2FC (fold change) ≥ 1.
All data were processed and normalized using MetaboAnalyst 5.0 (
10
Serum Metabolomic Analysis in Mice
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Serum collected at the end of the study period was used for non-targeted metabolomic data analysis. Serum was isolated from the whole blood collected from anesthetized mice via retro-orbital sampling. In accordance with the manufacturer’s instructions, the serum samples were placed on ice, three times the volume of ice-cold methanol was added, and the samples were centrifuged for 10 min (12,000 rpm, 4 °C). Then, the supernatant was collected and centrifuged for 5 min (12,000 rpm, 4 °C). Finally, the supernatant was collected and used for liquid chromatography with tandem mass spectrometry analysis (UPLC, Shim-pack UFLC SHIMADZU CBM A system, https://www.shimadzu.com/ 13 November 2022, Shimadzu, Columbia, OR, USA; MS, QTRAP® System, https://sciex.com/ 13 November 2022, Sciex, Framingham, MA, USA), according to standard protocols. The Metware Cloud Platform (Metware Biotechnology Co., Ltd., Wuhan, China) was used for metabolomics data analysis.
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