Coverage and characteristics of the E. coli spectral assay library. (a) Proteome coverage of the E. coli spectral assay library (complete library and library with top 6 ions and 100 variable windows applied) and SWATH-MS identified proteins with the developed library in comparison to the annotated reference proteome. (b) The graph depicts the number of E. coli peptides per protein in the SWATH assay library. (c) Retention time (RT) fit of +2 and +3 charge states of the same peptide in the assay library by DIALib-QC to assess the quality of the library. (d) Distribution of precursor m/z values across the acquired mass range in the assay library. (e) Frequency of precursor charge states observed in the assay library. (f) Frequency and type of peptide modification observed in the assay library. CAM: carbamidomethylation, Oxi: oxidation, PCm: S-carbamoylmethylcysteine, PGQ: pyroglutamate, and PGE: pyroglutamatic acid. (g) Distribution of peptide length in the assay library. (h) Distribution of the number of fragment ions per precursor. (i) Frequency of observed b- and y- ion fragments with CID fragmentation in the assay library.