Ethylene bridged hybrid c18

Labeled urine peptide samples were dissolved in 0.1% FA and separated with a Waters nanoAcquity UPLC before entering a Thermo Q-Exactive Orbitrap mass spectrometer (San Jose, CA). Each sample was injected twice. Mobile phase A consisted of water with 0.1% FA, and mobile phase B was composed of ACN with 0.1% FA. Samples were loaded onto a fabricated column with an integrated emitter. The 75 μm ID column was filled to a length of 15 cm using Ethylene Bridged Hybrid C₁₈ packing material (1.7 μm, 130 Å, Waters). Peptides were trapped onto the column in 100% A and separated using a solvent gradient of 0–10% B over 0.5 min and then 10–30% B over 70 min at a flow rate of 350 nL/min. Data-dependent acquisition (DDA) parameters recorded MS scans in profile mode from m/z 380–1500 at a resolution of 35K. Automatic gain control (AGC) targets of 1 x 10⁶ and maximum injection times (IT) of 100 ms were selected. The 15 most intense precursor ions were selected for MS² higher-energy collisional dissociation (HCD) fragmentation with an isolation width of 2.0 m/z and placed on an exclusion list for 40 s. Tandem mass spectra were acquired at a resolution of 17.5K in profile mode with an AGC target of 1 x 10⁵, a maximum IT of 150 ms, a normalized collision energy (NCE) of 27, and a fixed lower mass at m/z 110.

The LC-MS-based serum metabolic profiling adopted a Vanquish™ Horizon UHPLC system (Thermo Scientific, Germering, Germany) coupled with a Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer. The parameters of chromatography were as follows: column: Ethylene Bridged Hybrid C18 (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, Massachusetts, USA); gradient mobile phase: (A) deionized water containing 0.1% formic acid, (B) acetonitrile/isopropanol (1:1, v/v) mixture containing 0.1% formic acid; flow rate: 0.4 mL/min; sample injection volume: 2 μL; column temperature: 40 °C. The mobile phase gradient was: 0–3 min, A: 95–80%; 3–9 min, A: 80–5%; 9–13 min, A: 5–5%; 13–13.1 min, A: 5–95%; 13.1–16 min, A: 95–95%. The conditions of MS included the scan ranges (M/Z): 70–1050; Sheath gas flow rate (psi): 40; Aus gas flow rate (psi): 10; Aus gas heater temp (°C): 400; Normalized collision energy (V): 20–40–60; and IonSpray Voltage Floating (V): positive mode (ESI⁺), +3500; negative mode (ESI⁻), −2800. A QC sample was inserted for every 6 analytical samples during the experiment to evaluate the stability of the analytical system and assess the reliability of the results.

The LC-MS was performed on a Thermo UHPLC system equipped with a binary solvent delivery manager and a sample manager coupled to a Thermo Q Exactive Mass Spectrometer (Thermo Scientific, San Jose, CA, United States) equipped with an electrospray interface. The parameters of chromatography were as follows: column: Ethylene Bridged Hybrid C18 (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, United States); gradient mobile phase: (A) deionized water containing 0.1% formic acid, (B) acetonitrile/isopropanol (1:1, v/v) mixture containing 0.1% formic acid; flow rate: 0.4 ml/min; sample injection volume: 10 μL; column temperature: 40°C. The mobile phase gradient was: 0–3 min, A: 95–80%; 3–9 min, A: 80–5%; 9–13 min, A: 5–5%; 13–13.1 min, A: 5–95%; 13.1–16 min, A: 95–95%. The MS conditions included the scan ranges (M/Z): 70–1050; sheath gas flow rate (psi): 40; Aus gas flow rate (psi): 10; Aus gas heater temp (°C): 400; normalized collision energy (V): 20–40–60; and IonSpray Voltage Floating (V): positive mode (ESI⁺), +3500; negative mode (ESI⁻), −2800. A QC sample was inserted every 6 analytical samples during the experiment to evaluate the stability of the analytical system and assess the reliability of the results.