*2.8. LC-ESI-MRM*/*MS Analysis*

Liquid chromatography (LC) was performed on an Agilent 1290 Infinity UHPLC System with a reverse-phase ultra-high-performance LC (UHPLC) column (Agilent ZORBAX Eclipse Plus C18 Column, 95 Å, 2.1 mm i.d. × 100 mm, packed with 1.8 μm C18 resin) at a temperature of 50 ◦C. The mobile phases used in this study were 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The column was developed using a gradient of 0–2% solvent B for 5 min, 2–3% solvent B for 5 min, 3–50% solvent B for 10 min, 50–50% solvent B for 4 min, 50–0% solvent B for 1 min, and 0–0% solvent B for 9 min at a flow rate of 0.3 mL/min. The injected sample consisted of a mixture of digested plasma peptides (initial plasma volume: 40 μL) and isotope-labeled internal standard peptides. The UHPLC system was coupled to a triple quadrupole mass spectrometer (Agilent 6495 QQQ) by a standard-flow Jet Stream electrospray source operated in positive ion mode. Additional parameters included capillary voltage, 3.5 kV; nozzle voltage, 1 kV; gas temperature, 290 ◦C; drying gas flow rate, 11 L/min at 350 ◦C; nebulizer gas pressure, 40 PSI at 350 ◦C; and unit resolution for Q1 and Q3. MRM transitions were selected, and their collision energies optimized by Skyline (64-bit, version 19.1.0.193) software (Supplementary Table S4). The cell accelerator voltage was set to 5 V. Quantification experiments were performed using dynamic MRM (delta retention time: 3 min), with a total cycle time of approximately 1.5 s. The mass spectrometer was operated with MassHunter software (version B.08.00, Agilent), which generated MRM/MS data (\*.d). MRM results from extracted ion chromatograms were analyzed by Skyline and quantified relative to the corresponding stable isotope-labeled peptides (SpikeTides™; JPT Peptdie Technologies Berlin, Germany).
