We followed the protocol described previously (55). For Orbitrap Fusion Tribrid MS analysis, the tryptic peptides were purified with Pierce C18 spin columns (Thermo Fisher Scientific). Three micrograms of each fraction was autosampler loaded with a Thermo EASY nLC 1000 UPLC pump onto a vented Acclaim PepMap 100 (75 μm by 2 cm) nanoViper trap column coupled to a nanoViper analytical column (3 μm, 100 Å, C18, 0.075 mm, 500 mm; 164570, Thermo Fisher Scientific) with a stainless steel emitter tip assembled on the Nanospray Flex Ion Source with a spray voltage of 2000 V. Buffer A contained 94.785% H2O with 5% acetonitrile (ACN) and 0.125% formic acid (FA), and buffer B contained 99.875% ACN with 0.125% FA. The chromatographic run was 2 hours in total, with the following profiles: 0 to 7% for 3 min, 10% for 3 min, 25% for 80 min, 33% for 20 min, 50% for 3 min, 95% for 3 min, and again 95% for 8 min. Additional MS parameters include ion transfer tube temperature = 300°C, EASY-IC internal mass calibration, default charge state = 2, and cycle time = 3 s. Detector type was set to Orbitrap with a resolution of 60 K and a wide quad isolation [mass range = normal, scan range = 300 to 1500 mass/charge ratio (m/z), maximum injection time = 50 ms, automatic gain control (AGC) target = 200,000, microscans = 1, S-lens radio frequency level = 60, without source fragmentation, and data type = centroid]. Monoisotopic precursor selection was set as on, including charge states of 2 to 6 (reject unassigned). Dynamic exclusion was enabled with n = 1 for 30- and 45-s exclusion duration at 10 parts per million (ppm) for high and low (precursor selection decision = most intense, top 20, isolation window = 1.6, scan range = auto normal, first mass = 110, collision energy = 30%, collision-induced dissociation, detector type = ion trap, OT resolution = 30 K, IT scan rate = rapid, maximum injection time = 75 ms, AGC target = 10,000, Q = 0.25, and injected ions for all available parallelizable time).

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