The injection volume was set to 2 µL, except for the NIHNP library, which was set to 3 µL due to the lower concentration. H-ESI was used for ionization with a vaporizer temperature of 75°C and ion transfer tube temperature of 275°C. The voltages were set to 3000 V and 2000 V for positive and negative ionization modes, respectively. The sheath gas was set to 25 and aux gas was set to 5. No sweep gas was used. The MS n tree was built with the following main settings, with the Orbitrap as the mass analyzer: For MS , data were analyzed from m/z 115 to 2000 with a resolution of 30,000, an RF lens of 50%, an automatic gain control (AGC) target of 100% (40,000), and a maximum injection time (maxIT) of 50 ms. After one MS1 scan, the three most intense ions, with a minimum intensity of 6E5 in positive and 2E5 for negative ionization, were picked by data-dependent acquisition (DDA) with an isolation window of m/z 1.2, a resolution of 15,000, an AGC target of 30% (1,2E4), and maxIT of 50 ms for positive and 80 ms for negative. Three fragmentation experiments to cover different collision energies (a maximum of 9 scans) were conducted. For MS 2 , the energies were set to 20 eV and 60 eV, and the assisted collision energy to get the optimal MS 2 energy for further MS n stages from 15 eV, 30 eV, 45 eV, 60 eV, and 75 eV. From this assisted collision energy step, the top five signals, with a minimum intensity of 2E4 in positive and 1E4 in negative and within the mass range of m/z 90-2000 were isolated for MS with an MS isolation window of m/z 1.2 and an MS 2 isolation window of 2. The resolution was set to 60,000, the AGC target to 100% (5E4), and the maxIT to 200 ms for the positive and 500 ms for the negative ion mode. Three fixed collision energies of 20 eV, 40 eV, and 60 eV were applied, resulting in a maximum number of fifteen scans. The two most intense signals with a minimum intensity of 2E4 in positive and 1E4 in negative ion mode were picked from MS 3 scan with 40 eV for MS experiments, with an isolation window of m/z 2.2. All other settings were used as in MS . For MS , the two highest signals of an MS 4 scan with a collision energy of 40 eV and within a mass range of 150-2000 were further fragmented with an isolation window of m/z 3 and the same settings as MS and MS . Only 40 eV and 60 eV were used as the collision energy settings, as 20 eV showed mainly the precursor ion. Dynamic exclusion was carried out at every MS n stage, meaning that each precursor was picked three times within 200 s and was excluded for the following 70 s with a mass tolerance of m/z 0.2. Additionally, isotopes of selected precursor ions were excluded within a window of m/z 2 for unassigned isotopes. It needs to be noted that the maximum occurrence should be set to a number divisible by 3 to get experiments for all three collision energies. Prior to the standard analysis, multiple blank injections were analyzed, and the detected signals were set on a targeted mass exclusion list. This exclusion list was updated after running ten sample injections and the detection of reoccurring signals in all samples. The processing was done in mzmine. A full batch configuration file is supplied as Supplementary File 2 (mzmine_exclusion_blankprofile_pos.mzbatch) and Supplementary File 3 (mzmine_exclusion_blankprofile_neg.mzbatch). Our system showed higher background signals in empty scans or less rich fragmentation scans around m/z 149.72 and m/z 173.52 that were added to an exclusion list with a width of m/z 0.03 for all Ms n levels. The fragmentation tree is presented in Extended Data Fig. with an example shown in Supplementary Fig. . All settings can also be found in Supplementary Table and Supplementary Table .