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.
Uhplc qqq ms ms
Manufactured by Agilent Technologies
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The UHPLC-QQQ-MS/MS is an analytical instrument that combines ultra-high-performance liquid chromatography (UHPLC) with a triple quadrupole mass spectrometer (QQQ-MS/MS). It is designed for the sensitive and selective analysis of compounds in complex matrices.
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2 protocols using uhplc qqq ms ms
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Bacterial mRNA Quantification by UHPLC-QQQ-MS
2
Quantification of Oxidized Lipid Biomarkers
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The separation of F 4 -NeuroPs and F 2 -dihomo-IsoPs in the urine samples was performed by ultra high pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS; Agilent Technologies, Waldbronn, Germany), using the set-up described in ref. 22. Chromatographic separation was carried out on an ACQUITY BEH C 18 column (2.150 mm, 1.7 μm pore size) (Waters, MA, USA). The column temperatures were 6 °C (left) and 6 °C (right). The MRM was performed using the negative electrospray ionization (ESI) mode and the dwell time was 25 ms for all MRM transitions. The mobile phases were: (A) H 2 O containing 0.01% acetic acid (v/v) and (B) MeOH containing 0.01% acetic acid (v/v). The injection volume was 20 μL. The analysis time for each sample was 10.01 min. The flow rate was 0.2 mL min -1 , using a linear gradient scheme: (t; %B): (0.0; 60.00), (7.00; 70.00), (7.01; 90.00), (10.00; 90.00), (10.01; 60.00). The MS parameters fragmentor (ion optics) and collision energy were optimized for each compound. Data acquisition and processing were performed using Mass Hunter software version B.04.00 (Agilent Technologies, Waldbronn, Germany). The identification and quantification of F 4 -NeuroPs and F 2dihomo-IsoPs were carried out using the authentic markers previously described. 22
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