Genomic DNA (2 μg) was digested by nuclease P1, venom phosphodiesterase I (Type VI), and alkaline phosphatase (Sigma). After desalting and filtration, 10 μL of the solution was injected into HPLC-MS/MS for analysis. HPLC-MS/MS was carried out by reverse-phase ultra-performance liquid chromatography on an Agilent ZORBAX Eclipse XDB-C18 column (Rapid Resolution HT, 50 × 2.1 mm P.N. 927700-902), equipped with a ZORBAX Eclipse XDB-C8 guard column (Column: P.N. 821125-926, Cartridges P.N. 820555–901), eluted with buffer A (0.1% formic acid in H2O) and buffer B (0.1% formic acid in methanol) with a flow rate of 0.5 mL min-1 at 35°C with a 2% to 25% gradient in 4.5 min, with online mass spectrometry detection using Agilent 6410 triple-quadrupole (QQQ) LC mass spectrometer in multiple reaction monitoring (MRM) positive electrospray ionization (ESI) mode. The nucleosides were quantified using the nucleoside-to-base ion mass transitions of 258 to 142 with collision energy of 1 eV (hmC) and 228 to 112 with collision energy of 5 eV (C). Quantification and detection limits were determined by comparison with the standard curves obtained from nucleoside standards running at the same volume and time.
6410 qqq triple quadrupole lc mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States, Canada
The 6410 QQQ triple-quadrupole LC mass spectrometer is a laboratory equipment product manufactured by Agilent Technologies. It is designed to perform high-sensitivity, high-selectivity analysis of complex samples using liquid chromatography coupled with triple-quadrupole mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase, by Merck Group (7 mentions)
Nuclease p1, by Merck Group (6 mentions)
Nuclease p1, by Fujifilm (4 mentions)
C18 column, by Agilent Technologies (3 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (2 mentions)
0.22 μm filter, by Merck Group (2 mentions)
Nh4hco3, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ciap buffer, by Promega (1 mentions)
Uhplc qqq ms ms, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride filter, by Merck Group (1 mentions)
M6a rna methylation quantification kit, by Epigentek (1 mentions)
19 protocols using 6410 qqq triple quadrupole lc mass spectrometer
1
HPLC-MS/MS Analysis of DNA Methylation
2
Quantification of mRNA Nucleosides
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Poly(A) mRNA was isolated from total RNA using NEBNext poly(A) mRNA magnetic isolation according to the manufacturer’s protocol. One hundred fifty nanograms of mRNA was digested by nuclease P1 (1 U) in 25 μl of buffer containing 100 mM NH4OAc at 42 °C for 2 h. Subsequently, NH4HCO3 (1 M, 3 μl) and alkaline phosphatase (1 U) were added and further incubated for another 2 h at 37 °C. The samples were then filtered using a 0.22-μm PVDF filter (Millipore, P. N.: SLGVR04NL) and 5 μl of the solution was injected into the LC-MS/MS. HPLC-MS/MS was carried out by reverse-phase ultra-performance liquid chromatography on an Agilent ZORBAX Eclipse XDB-C18 column (Rapid Resolution HT, 50 × 2.1 mm (P.N. 927700-902), equipped with a ZORBAX Eclipse XDB-C8 guard column (Column: P.N. 821125-926, Cartridges P.N. 820555-901), eluted with buffer A (0.1% formic acid in H2O) and buffer B (0.1% formic acid in methanol) with a flow rate of 0.5 ml min−1 at 35 °C with a 2–25% gradient in 4.5 min, with online mass spectrometry detection using Agilent 6410 triple-quadrupole (QQQ) LC mass spectrometer in multiple reaction monitoring (MRM) positive electrospray ionization (ESI) mode. The nucleosides were quantified using the nucleoside to base ion mass transitions of 282.1 to 150.1 (m6A), 268.0 to 136.0 (A).
3
Quantification of m6A Modification in mRNA
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100–200 ng of mRNA was digested by nuclease P1 (2 U) in 25 μL of buffer containing 25 mM of NaCl, and 2.5 mM of ZnCl2 at 42 °C for 2 h, followed by the addition of NH4HCO3 (1 M, 3 μL) and alkaline phosphatase (0.5 U) and incubation at 37 °C for 2 h. The sample was then filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μL of the solution was injected into the LC-MS/MS. The nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column with online mass spectrometry detection using Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions of 282 to 150 (m6A), and 268 to 136 (A). Quantification was performed by comparison with a standard curve obtained from pure nucleoside standards run with the same batch of samples. The ratio of m6A to A was calculated based on the calibrated concentrations.
4
Quantification of m6A in Poly(A) RNA
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Total RNA of DC was isolated with Trizol reagent (Invitrogen), and mRNA was enriched by Dynabeads mRNA Purification Kit (Invitrogen). Removal of ribosomal RNA was confirmed by 2200 Tape Station detection (Agilent). In total, 2.5 μl of 10x Reaction Buffer (20 mM of ZnCl2, 100 mM of NaCl) and 1 μl of Nuclease P1 (1.2 U/μl) (Sigma) were added to 350 ng of purified mRNA and incubated at 37 ℃ for 2 h after adding H2O to a total volume of 2 μl. Then 2.5 μl of CIAP Buffer and 1 μl of CIAP (Promega) were added and incubated at 37 ℃ for another 2 h. The mix was diluted with H2O to 100 μl and filtered through a 0.22-µm filter (4 mm in diameter) (Nalgene) and then loaded to a C18 reverse-phase column coupled online to Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside to base on mass transitions of 268–136 (A), and 282–150 (m6A). A standard curve was obtained from pure nucleoside standards running at the same batch of samples. The m6A/A ratio in poly(A) RNA was quantified based on the calculated concentrations13 (link).
5
Quantification of m6A RNA Modification
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Double selected polyadenylated (poly(A)) mRNA was digested by nuclease P1 (1 U, Sigma) in 25 ml of buffer containing 20 mM NH4OAc (pH=5.3) at 37°C for 1 h, followed by an additional incubation with the addition of freshly made NH4HCO3 (1 M, 3 ml) and alkaline phosphatase (1 U, Sigma) at 37°C for 4 h. The samples were diluted to 50 ml and filtered (0.22-µm pore size, 4 mm diameter, Millipore), and 5 ml of the solution was injected for LC-MS/MS. Nucleosides were separated by reversed-phase ultra-performance liquid chromatography on a C18 column with online mass spectrometry detection by an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using retention time and nucleoside to base ion mass transitions of 282.1 to 150.1 (m6A), 268 to 136 (A), 284 to 152 (G), 245 to 113.1 (U), and 244 to 112 (C). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards run with the same batch of samples. The m6A level was calculated as the ratio of m6A to A based on the calibrated concentrations.
6
Quantification of m6A Modification in mRNA
7
Bacterial mRNA Quantification by UHPLC-QQQ-MS
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The highly purified bacterial mRNA was subjected to an UHPLC-QQQ-MS/MS (Agilent) analysis. Two hundred ng of mRNA or rRNA (on the beads of the mRNA Enrichment Kit) were digested by nuclease P1 (2 U) in 40 μl of nuclease buffer (25 mM of NaCl and 2.5 mM of ZnCl2) at 37°C for 2 h, followed by the addition of NH4HCO3 (1 M, 2 μl) and alkaline phosphatase (0.5 U) at 37°C for 2 h. The nucleosides were separated by reverse phase ultra-performance liquid chromatography by a C18 column on an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside-to-base ion mass transitions of 282 to 150 (m6A), 294 to 164 (m62A) and 268 to 136 (A). Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards. Three biological repeats have been performed for all bacterial strains.
8
Quantification of m6A Modification in RNA
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Total RNA from cell and tissue samples was extracted using TRIzol (Thermo Fisher scientific), from which mRNA was purified using the NEBNext Poly(A) mRNA magnetic separation module. In a 25 μL reaction system containing 10 mM NH4OAc (pH = 5.3), approximately 200 ng purified mRNA was incubated with nuclease P1 (0.5 U, Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 1 h at 42 °C, followed by the incubation with 3 μL NH4HCO3 and 1 μL alkaline phosphatase for 2 h at 37 °C. The samples were neutralized with 1 μL HCl, then diluted to 50 μL and filtered through a 0.22 μm filter (Millipore Corp, Billerica, MA, USA). All samples were separated by a C18 column using a reversed-phase ultra-high-performance-performance LC (Agilent Technologies, Santa Clara, CA, USA) and analyzed by an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer using positive ion electrospray ionization mode. All nucleosides were quantified by the transition from 268.0 to 136.0 (A) and 282.1 to 150.0 (m6A). Quantification was calculated using standard curves of standards run in the same batch, and m6A/A levels were calculated from calibration curves.
9
Quantitative Analysis of m6A in mRNA
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100–200 ng of mRNA was digested by nuclease P1 (2 U) in 25 μl of buffer containing 10 mM of NH4OAc (pH=5.3) at 42°C for 2 h, followed by the addition of NH4HCO3 (1 M, 3 μl, freshly made) and alkaline phosphatase (0.5 U). After an additional incubation at 37°C for 2 h, the sample was diluted to 50 μl and filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μl of the solution was injected into LC-MS/MS. Nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column with on-line mass spectrometry detection using an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside to base ion mass transitions of 282 to 150 (m6A), and 268 to 136 (A). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. The ratio of m6A to A was calculated based on the calibrated concentrations 9 .
10
mRNA Nucleoside Quantification by LC-MS/MS
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Two hundred nanograms of mRNA was digested with 1 U Nuclease P1 (Wako) in buffer containing 10% (v/v) 0.1 M CH3COONH4 (pH 5.3) at 42 °C for 3 h, followed by the addition of 1 U shrimp alkaline phosphatase (NEB) and 10% (v/v) Cutsmart buffer and incubated at 37 °C for 3 h. Then the sample was diluted to 50 μl and filtered through a 0.22 μm polyvinylidene difluoride filter (Millipore). Finally, 10 μl of the solution was used for LC-MS/MS. Nucleosides were separated using reverse-phase ultra-performance liquid chromatography on a C18 column coupled to online mass spectrometry detection using an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive ion mode. The nucleosides were quantified by comparison with the standard curve obtained from pure nucleoside standards run in the same batch as the samples. The ratio of m6A/A was calculated based on the calibration curves.
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