We implemented the automatic workflow for MS n tree library generation into mzmine and applied it to all seven compound libraries and both ionization polarities. The new core additions to allow for the handling of multi-stage fragmentation data include the 'MS n tree feature list builder', a set of advanced algorithms for spectral merging on multiple MS levels for various fragmentation energies, and the export of MS n tree libraries. Starting with the data import of converted open .mzML files or raw vendor formats, all spectra were denoised by removing signals below 2.5 times the lowest signal in each MS 1-n scan (see Supplementary Fig. ), accounting for the varying noise level due to ion trap-based normalization. This is illustrated in the Extended Data Fig. , where the lowest signal intensity increases with shorter filling times due to the normalization processing of the vendor. This fact prevents the use of an absolute noise level. In less intense MS n scans, broadly distributed noise signals were observed with an irregularly high mass defect for low m/z values (see Supplementary Fig. ).