Thermo ultimate 3000 uhplc system

LC separation was achieved with a Thermo Ultimate 3000 UHPLC system (Thermo Scientific) at a flow rate of 500 µl min^–1. Two different separation methods were applied. First separation was achieved by hydrophilic interaction (HILIC; Aquity UHPLC BEH Amide column [100 × 2.1 mm, 1.7 µm particle sizes; Waters]) as described in [54 ]. For HILIC analysis, 50 µl of the aqueous sample was dried (SpeedVac) and dissolved in MeCN. The C18 reversed phase (C18RP) separation was achieved using a Kinetex XB-C18 column (particle size 1.7 µm, pore size 100 Å; dimensions 50 × 2.1 mm², Phenomenex) as described elsewhere [55 (link)]. For mass analysis, LC instrument was coupled to a Thermo QExactive plus instrument (Thermo Fisher Scientific), and the mass spectrometer was operated both positive and negative FTMS mode at mass resolution of 30,000 (m/z = 400). Heated electro spray ionization (ESI) probe was used applying the following source parameters: vaporizer 350 °C; aux gas 5; ion spray voltage +3.5 kV, sheath gas, 50; sweep gas, 0; radio frequency level, 50.0; capillary temperature, 275 °C. To analyze the data, targeted extraction of ion chromatograms was conducted using emzed2 [56 (link)]. Retention time windows were determined based on chemical standards, and selected windows were normalized to background signal.

H₂S assay was based on a previously published method adapted here for tissue lysates [42 (link)]. First, approximately ~10–20 mg of the tissue samples were disrupted by a dismembrator. Alkylation/lysis was carried out by the addition of 500 µL PBS set to pH 8.0 containing 1 mM monobromobimane (Sigma Aldrich, St. Louis, MO, USA) in a light-protected environment. After a short sonication on ice the solutions were incubated for one hour at 37 °C in the dark. The reaction was stopped by the addition of 50 µL 50% TCA followed by centrifugation at 12,000× g 4 °C for 10 min to remove precipitated proteins. Supernatants were removed and transferred into HPLC vials for measurement, and the remaining pellets were redissolved in 300 µL 4% SDS/0.1 M NaOH for BCA protein assay. Bimane labeled species from the supernatants using 3 µL injection volumes were separated on a Phenomenex Luna C18(2) 250 × 2.0 mm × 3μm column on a Thermo Ultimate 3000 UHPLC system (Thermo Fisher, Waltham, MA USA). A linear gradient elution using solvents 0.1% TFA/H₂O (A) and 0.1% TFA/ACN (B) was carried out as described in Table 1. The fluorescent detector was set to excite at 390 nm and detect emission at 475 nm. Quantitation was conducted by establishing a calibration curve by derivatizing standardized H₂S solutions.