Bulk mRNA m6A modification quantification through LC-MS/MS was conducted by Wuhan Metware Co., Ltd. Briefly, 1 μg purified mRNA was sufficiently digested to nucleosides with S1 nuclease, phosphodiesterase, and alkaline phosphatase in 37 °C, then extracted by chloroform to get prepared solution samples. The samples were analyzed using a UPLC-ESI-MS/MS system (UPLC, ExionLCTM AD, https://sciex.com.cn/ ; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/ ). The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP) equipped with an ESI Turbo Ion-Spray interface, then operated in a positive ion mode and controlled by Analyst 1.6.3 software (Sciex). RNA modifications were analyzed using scheduled multiple reaction monitoring (MRM). Data acquisitions were performed using Analyst 1.6.3 software (Sciex). RNA modification contents were detected by MetWare (http://www.metware.cn/ ) based on the AB Sciex QTRAP 6500 LC-MS/MS platform.
Applied biosystems 6500 triple quadrupole
Manufactured by Thermo Fisher Scientific
The Applied Biosystems 6500 Triple Quadrupole is a high-performance mass spectrometry system designed for sensitive and selective quantitative analysis of small molecules. It features three quadrupole mass analyzers in tandem, enabling multiple-reaction monitoring (MRM) for accurate quantification of target analytes in complex matrices.
Automatically generated - may contain errors
5 protocols using applied biosystems 6500 triple quadrupole
1
mRNA m6A Quantification by LC-MS/MS
2
ABA and Transcriptome Analysis in Leaves
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After reaching the steady-state of photosynthesis, leaflets were harvested for ABA detection and transcriptome sequencing. Phytohormone contents were determined by a liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/ ; MS, Applied Biosystems 6500 Triple Quadrupole, www.appliedbiosystems.com.cn/ ). Briefly, the leaflets for photosynthetic measurements were harvested and frozen in liquid nitrogen. The samples were extracted with methanol/water/formic acid and filtered before LC-MS/MS analysis. The detailed protocol was described on MetWare (http://www.metware.cn/ ) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Samples were detected with three biological replicates.
3
Hormone Profiling of Grafted Tomato Stems
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The stem tissues of Z5/Z18-S, Z5/Z18-R, Z18/Z5-S, Z18/Z5-R, Z5/Z5, and Z18/Z18 were harvested 15 days before the Z5/Z18 and Z5/5 grafted plants matured. The samples were weighed and frozen in liquid nitrogen. IAA, ABA, GA, JA, SA, and ACC measurements were performed using an HPLC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHI-MADZU CBM30A system, www.shimadzu.com.cn/ (accessed on 6 December 2019); MS, Applied Biosystems 6500 Triple Quadrupole, www.appliedbiosystems.com.cn/ (accessed on 6 December 2019) at Wuhan Metware Biotechnology Co., Ltd. The mean hormone contents of the samples were compared using a two-tailed Student’s t-test with pooled variance.
4
Quantifying Endogenous Gibberellic Acids
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For measuring endogenous GAs contents, leaves of WT and OE-SlBES1.8 plants at 5-week-old were harvested and frozen into liquid nitrogen immediately. The measurement was conducted by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Briefly, leaf samples were ground using a mixer mill (MM 400, Retsch, Germany) to powder. GAs were extracted with 70% (v/v) acetonitrile from 500 mg powder. After adding the internal standards, 100 µL trimethylamine and 100 µL BPTAB, the resulting solution was vortexed, incubated at 90 for 1 hour, and evaporated to dryness under nitrogen gas stream followed by redissolving in 1 mL 70% (v/v) acetonitrile. The extracts were filtered through 0.22 µm filter and then analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLCTM AD, https://sciex.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). Finally, the output data was analyzed by Analyst 1.6.3 software.
5
Quantifying Endogenous Gibberellic Acids
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For measuring endogenous GAs contents, leaves of WT and OE-SlBES1.8 plants at 5-week-old were harvested and frozen into liquid nitrogen immediately. The measurement was conducted by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Briefly, leaf samples were ground using a mixer mill (MM 400, Retsch, Germany) to powder. GAs were extracted with 70% (v/v) acetonitrile from 500 mg powder. After adding the internal standards, 100 µL trimethylamine and 100 µL BPTAB, the resulting solution was vortexed, incubated at 90 for 1 hour, and evaporated to dryness under nitrogen gas stream followed by redissolving in 1 mL 70% (v/v) acetonitrile. The extracts were filtered through 0.22 µm filter and then analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLCTM AD, https://sciex.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). Finally, the output data was analyzed by Analyst 1.6.3 software.
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