4D-DIA Mass Spectrometry Analysis

4D-DIA mass spectrometry (MS) analysis was performed as the samples were separated using a C18 column (25 cm × 75 µm) on an EASY-nLC^TM 1200 system (Thermo Fisher, Waltham, MA, USA)^{60 (link),61 (link)}. The flow rate was 300 nL/min and the linear gradient was set accordingly. The DDA MS data for the library was acquired via the PASEF method as follows: MS data were collected over an m/z range of 100–1700, and during each MS/MS data collection, each TIMS cycle time was 1.1 s; each cycle included 1 MS and 10 MS/MS 100 msec TIMS scans; in each of the 10 PASEF MS/MS scans an average of 12 precursors were selected, resulting in an MS/MS data acquisition rate of 109 Hz. For the DIA, 56 DIA windows were acquired (automatic gain control target 3e6 and auto for injection time), and the collision energy was ramped linearly as a mobility function from 59 eV at 1/K0 = 1.6 Vs cm⁻² to 20 eV at 1/K0 = 0.6 Vs cm⁻². The MS/MS spectra were recorded from 100 to 1700 m/z.