10advp pumps

Samples were removed from the −80 °C freezer and allowed to warm to room temperature then vortexed for approximately 1 min before being placed into the autosampler and held at 24°C (Agilent 1100 series autosampler, Palo Alto, CA, USA) for LC/MS/MS analysis. Ten-twenty μL of eluants were injected separately for each sample to be rapidly separated using a C18 Zorbax reversed-phase analytical column to scan for individual compounds. Gradient elution (200 μL/min) then occurred, under the pressure created by two Shimadzu 10AdVP pumps (Columbia, MD, USA). Next, electrospray ionization was accomplished using an Applied Biosystems/MDS Sciex (Foster City, CA, USA) API3000 triple quadrupole mass spectrometer. A multiple reaction monitoring (MRM) setting on the LC/MS/MS was then used to analyze levels of each compound present in the sample injection. Synthetic standards were used to generate optimized MRM methods and standard curves for analysis.

Samples were removed from the −80°C freezer and allowed to warm to room temperature then vortexed for approximately 1 minute before being placed into the autosampler and held at 24°C (Agilent 1100 series autosampler, Palo Alto, CA) for the LC/MS/MS analysis. 20 μL of eluants were injected separately to be rapidly separated using a C18 Zorbax reversed-phase analytical column to scan for individual compounds. Gradient elution (200 μL/min) then occurred, under the pressure created by two Shimadzu 10AdVP pumps (Columbia, MD). Next, electrospray ionization was done using an Applied Biosystems/MDS SCIEX (Foster City, CA) API3000 triple quadrupole mass spectrometer. All compounds were analyzed using multiple reaction monitoring (MRM). Synthetic standards were used to generate optimized MRM methods and standard curves for analysis. We reported the MRM parent/fragment pairs previously (33 (link)), with the exception of phospho-LEA, which is MRM[−] 403.5/58.5. The mobile phases are also the same as we reported previously (33 (link)): mobile phase A, 80% HPLC-grade H₂O/20% HPLC-grade methanol, 1 mM ammonium acetate; mobile phase B, 100% HPLC-grade methanol, 1 mM ammonium acetate. Extreme outliers (10 times or higher than the average value in a group) were omitted from the analyses.