Steady-state levels of metabolites were measured as described before (64 ). Chromatographic separation was accomplished by hydrophobic interaction liquid chromatography using a Luna NH2 3 μm, 2 mm × 100 mm column, (Phenomenex) on a Vanquish Flex Quaternary UHPLC system (Thermo Fisher Scientific). For MS analyses an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) interfaced with an H-ESI, electrospray source (Thermo Fisher Scientific) was used. Data were collected for each sample in negative mode using two different mass ranges (70–700 and 220–900 m/z) to enhance sensitivity for larger less abundant compounds and in positive mode (70–900 m/z). The data were referenced to the MZCloud (mzcloud.org ) and Mass Bank (massbank.eu ) databases. All data were converted to MZXML format using MassMatrix. Peak areas, including isotopically enriched metabolites, were obtained using Mave for targeted analysis. Differential abundance of metabolites was computed in R using the student’s t test to determine significance and log2 fold change to determine magnitude and direction of change. Samples were quantile normalized using the R package preprocessCore before computing these metrics. FDR-corrected p values were also computed to adjust for multiple-testing bias.
Vanquish flex quaternary uhplc system
Manufactured by Thermo Fisher Scientific
The Vanquish Flex Quaternary UHPLC system is a high-performance liquid chromatography (HPLC) instrument designed for efficient and reliable separation and analysis of a wide range of sample types. It features a quaternary pump for precise solvent delivery and advanced data acquisition capabilities for high-resolution chromatographic results.
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Lab products found in correlation
2 protocols using vanquish flex quaternary uhplc system
1
Metabolite Profiling via HPLC-MS/MS
2
Quantification of m6A in Poly(A) RNA
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For quantification of m6A in poly(A) RNA, nucleoside digestion was performed as previously described(31 (link)). Separation was accomplished by reversed phase chromatography with an Acquity UPLC HSS T3 (Waters) on a Vanquish™ Flex Quaternary UHPLC system (Thermo Fisher Scientific). Mass spectrometry was performed on a Quantiva™ triple quadrupole mass spectrometer interfaced with an H-ESI electrospray source (Thermo Fisher Scientific). Data were analyzed with Tracefinder 4.1 (Thermo Fisher Scientific) and Qual browser of Xcalibur 3.0. The mass transitions (precursor → product) for m6A were 282 → 94, 282 → 123 and 282 → 150.
Changes in m6A were also measured by dot blot from 50ng of poly(A) RNA. Blotting was performed as previously described for 5-hydromethylcytosine(32 (link), 33 (link)), except that Diagenode C15410208 (1:400) was used as primary antibody.
Changes in m6A were also measured by dot blot from 50ng of poly(A) RNA. Blotting was performed as previously described for 5-hydromethylcytosine(32 (link), 33 (link)), except that Diagenode C15410208 (1:400) was used as primary antibody.
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