Comprehensive Metabolomic and Proteomic Analysis

Metabolome analyses were performed on a Thermo Ultimate RS 3000 coupled online to a Q-Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization probe (HESI II). The MS was calibrated by Thermo calmix (Pierce™ calibration solutions in both polarities. Lipidome analysis was performed on an Ultimate RS 3000 UHPLC (Thermo Fisher), coupled online to a TimsTOF Pro Quadrupole Time of Flight (Q-TOF) (Bruker Daltonics, Bremen, Germany) equipped with an Apollo II electrospray ionization (ESI) probe. Proteomics analysis used a nanoLC coupled to a tribrid Orbitrap Lumos (Thermo Fisher Scientific, Bremen). Methods were performed as described previously [27 (link)–29 (link)]. Detailed mass spectrometry conditions and raw data alignment, filtering, and annotation parameters are described in (Additional file 1). For the assessment of repeatability and instrument stability over time, a QC strategy was applied. A quality control (QC) sample was prepared by pooling the same aliquot (10 µL) from each sample. Samples were injected in randomized order, and blank samples were injected regularly and used to assess carryover and exclude background signals.