Plasma Metabolomic Profiles Reveal Site-Specific Effects of Estrogen Receptor Alpha Phosphorylation in Mice

(Baltimore, MD, USA) for metabolomic analysis. Metabolites were extracted from plasma samples across all experimental groups using 100% liquid chromatography-mass spectrometry (LC-MS) grade methanol, adhering to an 80:20 methanol-to-sample (vol/vol) ratio as described in previous studies. Following extraction, methanol and water were evaporated from the samples, leaving behind dried metabolites, which were subsequently reconstituted in a solution of 50% acetonitrile (vol/vol) in MS-grade water for metabolomic analysis.

For data acquisition, a Thermo Scientific IQX Mass Spectrometer was used in conjunction with a Vanquish UPHLC system. The samples were maintained at 4°C throughout the LC process, and an injection volume of 2 μL was utilized for each sample. A 15-minute reverse-phase chromatography protocol was applied using a Discovery HSF5 reverse-phase column (Sigma).

The mobile phase consisted of 0.1% formic acid in MS-grade water for the aqueous phase and 0.1% formic acid in acetonitrile for the organic phase. Prior to data acquisition, instrument calibration was performed to ensure optimal sensitivity and accuracy. The final metabolite intensity values were obtained by integrating chromatographic peak areas, with normalization performed relative to the sample protein concentration.The primary tabular file (ERalpha_phospho_metabolomics.xlsx) contains quantified plasma metabolites (rows) measured across 18 biological samples (columns).