For the purine and pyrimidine analysis, we operated a SCIEX 5500 Triple-Quadrupole LC-MS mass spectrometer fitted with a Turbo V ion source, online connected to an ultra-high performance liquid chromatography Agilent 1260 UHPLC system. Analyst v1.6.1 (SCIEX) was used for all SRM data acquisition, the development of the HPLC method, and the optimization of analyte-specific SRM transitions. Skyline-daily version 4.2.1.19004 was used for LC-SRM-MS data analysis and processing.
For the purine and pyrimidine analysis, urine from five mice was collected from voluntary expulsion, and 20 μL aliquots were stored at −80 °C until ready for analysis. Urine aliquots were thawed on ice and 80 μL of methanol was added containing 2-chloroadenosine (IS) at a final concentration of 2.5 μM to each 20 μL urine aliquot. The mixture was vortexed vigorously for ~30 seconds and protein precipitation was completed by incubating at −20 °C for 30 min. After this, samples were vortexed vigorously for ~30 seconds and centrifuged at 15,000 rpm for 10 min at 4 °C. An 80 μL aliquot of the supernatant was carefully removed without disturbing the pellets and transferred to an HPLC autosampler vial fitted with inserts; 2 μL were injected per HPLC-SRM-MS analysis.
Synthetic standards for compounds indicated inTable S1 were obtained from IROA (Mass Spectrometry Metabolite Library of Standards, MSMLS) or Sigma-Aldrich, St. Louis, MO. 100 μM stocks were prepared in 80% methanol and stored in −80 °C prior to use. A final standard mixture of all compounds at 5 μM (containing the internal standard/IS, 2-chloroadenosine at 2.5 μM), was prepared prior to analysis and injected at the onset of each set biological sample set. The Skyline document for urine purine/pyrimidine analysis has been uploaded to Panorama Public at https://panoramaweb.org/SkylineForSmallMolecules.url .
For the purine and pyrimidine analysis, urine from five mice was collected from voluntary expulsion, and 20 μL aliquots were stored at −80 °C until ready for analysis. Urine aliquots were thawed on ice and 80 μL of methanol was added containing 2-chloroadenosine (IS) at a final concentration of 2.5 μM to each 20 μL urine aliquot. The mixture was vortexed vigorously for ~30 seconds and protein precipitation was completed by incubating at −20 °C for 30 min. After this, samples were vortexed vigorously for ~30 seconds and centrifuged at 15,000 rpm for 10 min at 4 °C. An 80 μL aliquot of the supernatant was carefully removed without disturbing the pellets and transferred to an HPLC autosampler vial fitted with inserts; 2 μL were injected per HPLC-SRM-MS analysis.
Synthetic standards for compounds indicated in
