Thermo xcalibur 4

As previously reported [53 (link)], serum samples were analyzed with UHPLC–HRMS. A binary solvent system consisting of (A) 0.1% formic acid (HCOOH) in acetonitrile (CH₃CN) and (B) 0.1% v/v HCOOH was used for chromatographic analysis of polar phenolics, along with an Accucore™ column (C18, 150 × 2.1 mm, 2.6 μm, Thermo Fisher Scientific, Waltham, MA, USA). Gradient elution began with 5% of solvent B for 1.1 min, followed by a rapid increase to 25% in 1.1 min, which then remained isocratic for an additional 1.1 min. After 0.6 min, the concentration of B was raised to 40% and remained at this level for 2.6 min. In the next 3 min, the concentration of B increased to 95%. The initial conditions were then restored in 0.4 min and maintained for 5.6 min for column equilibration, which resulted in a total run time of 18 min; the injection volume was 5 μL. HRMS data were acquired with an Exactive Plus™ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), which operated in negative heated electrospray ionization mode (H-ESI). Thermo XCalibur 4.0 was used to handle acquisition, while quantitative results were obtained with TraceFinder™ 4.1 (Thermo Fisher Scientific, San Jose, CA, USA).

Ten microliters (μl) of each fraction were analyzed by Q Exactive (Thermo, USA) mass spectrometer coupled to a Proxeon Biosystem Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, USA) in the LC-MS experiments. The peptide mixture (5 g) was loaded onto a C18 column (75 μm × 25 cm, Thermo,USA) packed with RP-C18 (5 m) resin in buffer A (2% ACN with 0.1% formic acid), and eluted with a linear gradient of buffer B (80% ACN with 0.1% formic acid) at a flow rate of 300 nl/min for 120 min using IntelliFlow technology. The equate underwent electrospray ionization for LC-MS analysis. The MS/MS instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top-20 method based on the selection of the most abundant precursor ions from the survey scan (350–1300 m/z) for HCD fragmentation. Determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was 18 s. Survey scans were acquired at a resolution of 70,000 at m/z 200, and the resolution for the HCD spectra was set to 17,500 at m/z 200. The normalized collision energy was 30 eV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. Thermo Xcalibur 4.0 (Thermo, USA) was used to collect MS analysis data via DDA mode.

A Thermo Q Exactive Orbitrap hybrid mass spectrometer (Thermo Fisher, Waltham, MA, USA) was equipped with an ESI source. The samples were measured in both positive and negative ionization modes separately. The capillary temperature was 320 °C and spray voltages were 4.0 kV in positive ionization mode and 3.8 kV in negative ionization mode, respectively. The resolution was 35,000 for MS1 scans and 17,500 for MS2 scans. The scanned mass interval was 100–1500 m/z. For the tandem MS (MS/MS) scans, the collision energy was set to 30 nominal collision energy units. The difference between measured and calculated molecular ion masses was less than 5 ppm in each case. The data were acquired and processed using Thermo Trace Finder 2.1 software based on own and internet databases (Metlin, Mass Bank of North America, m/z Cloud). After processing, the results were manually checked using Thermo Xcalibur 4.0 software (ThermoFisher, Waltham, MA, USA).