Peptides were separated with NANO HPLC Capillary Column (Nikkyo Technos, Japan), with elution performed over 90 min with a gradient of 5% to 95% acetonitrile at 500 nl/min. Samples were analyzed with Orbitrap Exploris 240 (Thermo Fisher Scientific) via nano-electrospray ionization. The instrument was operated in data-dependent acquisition mode, capturing full-scan MS spectra at a resolution of 60,000. The top 10 peaks were fragmented with a 30% collision energy and 1.6 m/z isolation window, with auto dynamic exclusion.