LC-MS/MS quantification of m6A was performed by Cloudseq Biotech Inc. (Shanghai, China) following the vendors recommended protocol. Total RNA was isolated using TRIzol reagent (Catalog No. 15596026, Invitrogen) following to the manufacturer’s instruction. In brief, 1 µg of total RNA was digested by 4-µl nuclease P1 (Catalog No. N8630, Sigma, St. Louis, MO) in 40-µl buffer solution (10 mM Tris-HCl pH 7.0, 100 mM NaCl, 2.5 mM ZnCl2) at 37 °C for 12 h, followed by incubating with 1-µl alkaline phosphatase (Catalog No. P5931, Sigma) at 37 °C for 2 h. RNA solution was diluted to 100 µl and injected into LC-MS/MS. The nucleosides were separated by reverse phase high-performance liquid chromatography on an Agilent C18 column (Catalog No. 5188–5328, Agilent Technologies, San Diego, CA), coupled with MS detection using AB SCIEX QTRAP 5500 (Catalog No. AB SCIEX QTRAP 5500, AB Sciex LLC, Framingham, MA). Pure nucleosides were used to generate standard curves, from which the concentrations of adenosine (A) and m6A in the sample were calculated. The level of m6A was then calculated as a percentage of total unmodified A.
Qtrap 5500
Manufactured by AB Sciex
Sourced in United States, Japan, Germany, Canada, United Kingdom, Denmark, France, Austria
The QTRAP 5500 is a high-performance hybrid triple quadrupole linear ion trap mass spectrometer. It combines the quantitative capabilities of triple quadrupole technology with the qualitative power of a linear ion trap, enabling both quantitative and qualitative analysis in a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Analyst 1, by AB Sciex (23 mentions)
1290 hplc, by Agilent Technologies (13 mentions)
Lc 20ad hplc, by Shimadzu (12 mentions)
Multiquant, by AB Sciex (11 mentions)
Nexera x2, by Shimadzu (11 mentions)
Acquity uplc beh c18 column, by Waters Corporation (10 mentions)
Analyst software, by AB Sciex (10 mentions)
Multiquant 3, by AB Sciex (8 mentions)
Multiquant software, by AB Sciex (8 mentions)
Uflc xr, by Shimadzu (7 mentions)
Triversa nanomate, by Advion (6 mentions)
Formic acid, by Thermo Fisher Scientific (6 mentions)
Lc 30 ultrahigh performance liquid chromatography system, by Shimadzu (5 mentions)
C18 hplc column, by Agilent Technologies (5 mentions)
Lipidview software, by AB Sciex (5 mentions)
Beh c18 column, by Waters Corporation (5 mentions)
Ultra performance liquid chromatography (uplc), by Waters Corporation (5 mentions)
Absoluteidq p180 kit, by Biocrates (5 mentions)
Sil 20ac autoinjector, by Shimadzu (5 mentions)
Eclipse plus c18 column, by Agilent Technologies (5 mentions)
509 protocols using qtrap 5500
1
Quantification of m6A by LC-MS/MS
2
Quantitative AP-MALDI Mass Spectrometry Analysis
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The quantitative experiments were performed on an AB SCIEX QTRAP 5500 (SCIEX, Framingham, MA, USA) mass spectrometer coupled to an AP-MALDI ion source (MassTech, Inc., Columbia, MD, USA) equipped with a 355 nm Nd: YAG laser source.
The AB SCIEX QTRAP 5500 system was operated in SRM with positive ion polarity mode. Analyst software 1.7.1 (SCIEX) was used for data acquisition and parameter optimization for SRM. For SRM analysis, ion spray voltage was optimized to 4000 V, interface heater temperature was set at 220 C, and declustering potential (DP) was set to 91 V for best signals. The collision energy (CE) was optimized to 33 eV for 273.040 u and 45 eV for 229.007 u to achieve fragmentation. The laser energy of the AP-MALDI ion source was optimized at 80% for the analysis with the Target (MassTech, Inc., Columbia, MD) software. AP-MALDI quantitation sequence was run automatically with the Target software. The obtained AP-MALDI SRM data were qualitatively interpreted with PeakView 2.2 (SCIEX). PeakView 2.2 uses the Analyst-generated raw data as an input file and gives a text file as an output. The output text files contain the intensities of the chosen SRM transitions of a particular AP-MALDI spot(s)/well(s). These text files were imported in Microsoft Excel, and the quantitative graphs were plotted with Microsoft Excel.
The AB SCIEX QTRAP 5500 system was operated in SRM with positive ion polarity mode. Analyst software 1.7.1 (SCIEX) was used for data acquisition and parameter optimization for SRM. For SRM analysis, ion spray voltage was optimized to 4000 V, interface heater temperature was set at 220 C, and declustering potential (DP) was set to 91 V for best signals. The collision energy (CE) was optimized to 33 eV for 273.040 u and 45 eV for 229.007 u to achieve fragmentation. The laser energy of the AP-MALDI ion source was optimized at 80% for the analysis with the Target (MassTech, Inc., Columbia, MD) software. AP-MALDI quantitation sequence was run automatically with the Target software. The obtained AP-MALDI SRM data were qualitatively interpreted with PeakView 2.2 (SCIEX). PeakView 2.2 uses the Analyst-generated raw data as an input file and gives a text file as an output. The output text files contain the intensities of the chosen SRM transitions of a particular AP-MALDI spot(s)/well(s). These text files were imported in Microsoft Excel, and the quantitative graphs were plotted with Microsoft Excel.
3
Shotgun Lipidomic Analysis of Phospholipids
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The species of all phospholipids, SM, DAG and CE were analyzed by shotgun analysis on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB SCIEX) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences) [59 ]. These analyses were performed using both positive and negative ion modes using multiple precursor ion scanning (MPIS) and neutral loss (NL) based methods [56 (link), 60 (link)], whereas CEs were analyzed in positive ion mode [61 (link)]. Sphingolipids were analyzed by reverse phase ultra-high pressure liquid chromatography (UHPLC) as previously described [62 (link)] using an Acquity BEH C18, 2.1×50 mm column with a particle size of 1.7 μm (Waters, Milford) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB SCIEX). A 25 min gradient using 10 mM ammonium acetate in water with 0.1% (v/v) formic acid (mobile phase A) and 10 mM ammonium acetate in 4:3 (v/v) acetonitrile:2-propanol containing 0.1% (v/v) formic acid (mobile phase B) was used. Quantification of sphingolipids was performed using multiple reaction monitoring. Data from one such experiment are shown; similar results were obtained in another independent experiment.
4
Comprehensive Metabolite Profiling Protocol
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The methods for measuring blood metabolites, including amino acids, hormones, vitamins, microelements and heavy metals, were described in detail by Jie et al.67 (link) Briefly, we detected the amino acids from 40 μL plasma using ultra-high pressure liquid chromatography (UHPLC) coupled to an AB Sciex Qtrap 5,500 mass spectrometer (MS) (AB Sciex, US) with an electrospray ionization (ESI) source; to measure hormones, 250 μL plasma was used and detected via UHPLC-MS (AB Sciex Qtrap 5,500) with an atmospheric pressure chemical ionization (APCI) source; water-soluble vitamins were detected from 200 μL plasma via UPLC-MS (Waters Xevo TQ-S Triple Quad, Waters, US) with an ESI source; and fat-soluble vitamins were detected from 250 μL plasma via UPLC-MS (AB Sciex Qtrap 4,500, AB Sciex, US) with an APCI source; we detected the microelements and heavy metals in 200 μL whole blood via an Agilent 7,700x ICP-MS (Agilent Technologies, Japan) equipped with an octupole reaction system (ORS). UPLC-MS was used in positive ion mode. All measured items are shown in Table S1 .
5
Quantifying Phosphoinositide Levels in MDCK Cells
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Phosphoinositide quantification was performed as described previously (Haag et al., 2012 (link)). Briefly, MDCK cells were plated in 6-well plates and grown for 5 days in Matrigel. Cells from two 6-well plates were combined and subjected to an acidic–neutral extraction of trichloroacetic acid (TCA)-washed cell pellets using PI(4)P 17:0–20:4 and PI(4,5)P2 17:0–20:4 from Avanti Polar Lipids as internal standards for quantification. Mass spectrometry was performed on a QTRAP 5500 instrument (AB Sciex) equipped with a Triversa NanoMate system (Advion Biosciences). Phosphoinositides were measured by scanning for neutral losses of m/z 357 (phosphatidylinositol) and m/z 437 (PIP2) on an AB Sciex QTRAP 5500 instrument at collision energies of 25 eV and 35 eV, respectively. Mass spectra were evaluated using LipidView software (AB Sciex). PIP and PIP2 amounts were normalized to total phospholipids, which was determined in neutral and acidic phases, as described previously (Özbalci et al., 2013 (link)).
6
Quantification of Anthocyanins in ABE Using LC-MS/MS
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According to a previous method with modification, the individual anthocyanin in ABE was quantified using LC-MS/MS system [31 (link),32 (link)]. The high-performance liquid chromatography (HPLC) system (Agilent 1290, Agilent Technologies, Santa Clara, CA, USA) consisted of a binary pump and autosampler with a reverse-phase C-18 Inertsil ODS-2 column (250 × 4.6 mm, 5 µm, 150 A°, Techno Quartz Inc., Tokyo, Japan). The mobile phase was formic acid: water (10:90 v/v; mobile phase A) and formic acid: water: acetonitrile: methanol (10:40:22.5:22.5 v/v/v/v; mobile phase B). The anthocyanin was separated by a linear gradient following: 15–20% (B) in 5 min, 20–27% (B) in 35 min, 27–65% (B) in 45 min, 65–100% (B) in 50 min and then back to 15% (B) until 60 min at a flow rate of 0.6 mL/min. The injection volume of the sample was 5 µL. The anthocyanin was quantified using a mass spectrophotometer with electrospray ionization (ABSciex QTRAP 5500, Sciex, Framingham, MA, USA). The anthocyanin content was determined by the multiple reaction monitoring-enhanced product ion mode (MRM-EPI) under condition was set as follows: ion spray voltage 5.5 kV, source temperature 500 °C, curtain gas 25 psi, collision energy 20 eV. Data were analyzed using “ABSciex analyst” software (Sciex, Framingham, MA, USA). Anthocyanins standard was cyanidin-3-glucoside (C3G) and delphinidin-3-glucoside (D3G).
7
LC-MS/MS Quantification of Analytes
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LC-MS/MS system comprised of liquid chromatography (Shimadzu, MD, USA) with a binary pump (LC-30AD), an autosampler (SIL-30AC), a controller (CBM-20A), a degasser (DGU-20A5R), a column oven (CTO-20A), and an ABSciex QTRAP 5500 mass spectrometer (SCIEX, Foster City, CA, USA) with electron spray ionization (ESI) source. The chromatograms were monitored using Analyst 1.7 software, and the data were analyzed in MultiQuant 3.0 software (SCIEX, Foster City, CA, USA). The analytes were separated using Synergi™ Fusion-RP column (2.5 µm, 100 × 2 mm) obtained from Phenomenex (Torrance, CA, USA). All the analyses were performed in positive ion mode.
8
Quantifying IAA via LC-MS
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IAA was quanti ed by liquid chromatography-mass spectrometry (LC-MS), essentially as described [44] . Brie y, 50 mg of frozen tissue was homogenized in 500 μL of extraction buffer (isopropanol: double distilled water: acetic acid= 2:1:0.002, vol/vol/vol). After shaking on ice for 30 min, 1 mL dichloromethane was added to each sample and shaken for another 30 min. Then, samples were centrifuged at 13,000 g for 5 min at 4 °C; the lower phase was dried under a stream of nitrogen gas, and redissolved with methyl alcohol. Each sample solution was injected into the reverse-phase C 18 HPLC column (2.1mm × 100 mm, 2.7 μm, Agilent, USA) for LC-MS (Shimadzu LC-30 ultrahigh performance liquid chromatography system, Shimadzu, Japan; AB Sciex QTRAP 5500, SCIEX, USA) analysis. An IAA standard (≥98%, 45533, Sigma, USA) was used as the external standard.
9
Quantification of IAA by LC-MS
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IAA was quanti ed by liquid chromatography-mass spectrometry (LC-MS), essentially as described [41] . Brie y, 50 mg of frozen tissue was homogenized in 500 µL of extraction buffer (isopropanol: double distilled water: acetic acid = 2:1:0.002, vol/vol/vol). After shaking on ice for 30 min, 1 mL dichloromethane was added to each sample and shaken for another 30 min. Then, samples were centrifuged at 13,000 g for 5 min at 4 °C; the lower phase was dried under a stream of nitrogen gas, and redissolved with methyl alcohol. Each sample solution was injected into the reverse-phase C 18 HPLC column (2.1 mm × 100 mm, 2.7 µm, Agilent, USA) for LC-MS (Shimadzu LC-30 ultrahigh performance liquid chromatography system, Shimadzu, Japan; AB Sciex QTRAP 5500, SCIEX, USA) analysis. An IAA standard (≥ 98%, 45533, Sigma, USA) was used as the external standard.
10
Quantifying IAA levels via LC-MS
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IAA was quanti ed by liquid chromatography-mass spectrometry (LC-MS), essentially as described [55] . Brie y, 50 mg of frozen tissue was homogenized in 500 μL of extraction buffer (isopropanol: double distilled water: acetic acid= 2:1:0.002, vol/vol/vol). After shaking on ice for 30 min, 1 mL dichloromethane was added to each sample and shaken for another 30 min. Then, samples were centrifuged at 13,000 g for 5 min at 4 °C; the lower phase was dried under a stream of nitrogen gas, and redissolved with methyl alcohol. Each sample solution was injected into the reverse-phase C 18 HPLC column (2.1mm × 100 mm, 2.7 μm, Agilent, USA) for LC-MS (Shimadzu LC-30 ultrahigh performance liquid chromatography system, Shimadzu, Japan; AB Sciex QTRAP 5500, SCIEX, USA) analysis. An IAA standard (≥98%, 45533, Sigma, USA) was used as the external standard.
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