In Method 2, the AGC target was increased to 100% (50k). In Method 3, the mass resolution was increased from 15k to 60k because the 128 ms scan time needed for 60k utilizes the 200 ms maximum injection time optimally. The comparison of all methods is summarized in Extended Data Fig. by plotting the lowest-intensity signal, here defined as the noise level, of each MS 3 spectrum against its corresponding 1/injection time. A higher AGC target shifts the scan rate to longer injection times, resulting in lower noise levels. This can be explained by the internal processing in trap-based MS instruments, where measured intensities are divided by the injection time for response normalization. Similar trends toward even lower noise levels were achieved by increasing the mass resolution, which lowers the slope of the linear regression (Method 3, magenta). Overall, Method 3 resulted in higher signal-to-noise (SN) ratios, as exemplified in Supplementary Fig. , which compares all MS 3 spectra (several MS 3 precursors) of the same MS 2 precursor ion picked in all three methods. Here, Method 3 halved the noise level to 1E5, offering a better distinction between signals and noise. During data processing, mass detection in mzmine applied denoising with a threshold of 2.5 times the lowest signal in each scan to clean the spectra before exporting with our automatic library building workflow. A histogram in Extended Data Fig. compares the number of signals in individual scans for all exported MS scans to measure spectral quality. Overall, the higher AGC and mass resolution yielded more spectra with more than ten fragment signals and increased the average number of MS 3 signals from 14 in Method 1 to 19 and 23 in Method 2 and 3, respectively.