Qtrap 4500 mass spectrometer

Around 0.5 $\mathrm{\mu }\mathrm{L}$ of cell suspension was transferred to a nanospray capillary. A DC voltage of −1.3 kV was applied via a stainless-steel electrode for several seconds to promote electro-migration. After solvent evaporation, a droplet (~50 μL) of assistant solvent (methanol:acetonitrile = 1:1 (v/v) with 1% formic acid) was applied to the capillary tip. Meanwhile, a voltage of +1.8 kV was applied to initiate on-demand MS analysis. For PB-MS/MS analysis, multiple droplets were dropped sequentially to the nanoESI tip to analyze different lipid species. It took 1~2 min to transfer a single cell to the capillary and perform electro-migration, 3~5 min to evaporate the liquid in the tip, and 4~6 min for multi-rounds sampling and data acquisition. For lipid sum compositions analysis, a droplet of 10 mM LiCl in methanol:acetonitrile = 1:1(v/v) and a voltage of +1.5 kV were applied for DAESI. For negative MS/MS analysis to confirm fatty acyl-chains, a droplet of 50 mM ammonium acetate solvent in methanol:acetonitrile:water = 4.5:4.5:1 (v/v) and a voltage of −1.8 kV were applied for DAESI. For sn-position identification, a droplet of 50 mM ammonium bicarbonate solvent in methanol:acetonitrile:water = 4.5:4.5:1 (v/v) and a voltage of −1.8 kV were applied for DAESI. All MS analysis was performed by QTRAP 4500 mass spectrometer (Sciex, Toronto, CA).

Serum levels of taurine and spermine were determined with the AbsoluteIDQ™ p180 kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) using the flow injection analysis tandem mass spectrometry (FIA-MS/MS) as well as liquid chromatography (LC-MS/MS) technique on a QTRAP 4500 mass-spectrometer (Sciex, USA). All measurements were performed as described in the manufacturer's kit manual. Identification and quantification of the metabolites were achieved using multiple reaction monitoring (MRM) along with internal standards. Calculations of metabolite concentrations were automatically performed by MetIDQ™ software (BIOCRATES Life Sciences AG). The validation of metabolomic and cytokine/growth factor measurements are presented in the Supplementary Material section.

Polyphenolic profile was determined according to [19 (link)] by a Nexera XR UHPLC system (Shimadzu, Tokyo, Japan) coupled to a Qtrap 4500 mass spectrometer (Sciex, Toronto, ON, Canada) equipped with a heated ESI source (V-source). The analytes were separated using an Excel 2 C18-PFP column (10 cm × 2.1 mm ID) from ACE (Aberdeen, UK) packed with 2 μm particles and equipped with a security guard. The mobile phases were 1% acetic acid in water (A) and acetonitrile (ACN) (B). The flow rate was set at 0.300 mL min⁻¹ for a total run of 15 min. The acquisition and quantification of ion currents was performed in MRM mode. Standard compounds, namely, tyrosol, hydroxytyrosol, diosmetin, luteolin, apigenin, oleacein and oleocanthal, were purchased from Sigma-Aldrich (Milan, Italy). Data collection and processing were performed with Analyst 1.6.2 software and quantification with Multiquant 3.0 software (Sciex). The selected ions, together with the main HPLC-MS/MS parameters are reported in Table S2 (Supplementary Material).