(A) Workflow. (A1) HeLa cells were synchronized in S-phase using thymidine (dark orange). Upon release from thymidine block, cells were treated with nocodazole and samples were collected after 4 hr (medium dark orange), 8 hr (orange), and 10 hr (light orange). (A2) APC/C was co-immunoprecipitated using an anti-CDC27 antibody. (A3) All samples were trypsinized separately and analyzed by LC-MS/MS in DDA mode to generate a spectral library, which was subsequently analyzed by SWATH-MS (see Supplementary file 1 for raw peptide intensities of all quantified APC/C proteins). (A4) FLEXIQuant-LF-based differential modification analysis of APC/C proteins (see Supplementary file 2 for all resulting RM scores). (B) FLEXIQuant-LF overview. (B1) Firstly, intensities of unmodified peptides are used to indirectly identify and quantify the modification extent of a protein using the following steps. (B2) A RANSAC-based robust linear regression model is fitted to the intensities of unmodified peptide species using a reference sample as independent variable and the sample of interest as dependent variable and the vertical distance to the regression line of each peptide is determined. (B3) For each peptide, the distance is then normalized by dividing by the slope of the regression line multiplied by the intensity of the peptide in the reference sample and the result is subtracted from one to yield raw scores. (B4) Peptides with a raw score above three standard deviations of the median (MAD) of all raw scores are classified as outliers and excluded from the subsequent RM score calculation. The remaining raw scores are then scaled using the median of the three highest raw scores resulting in a metric, termed RM score which is equal to one minus the extent of modification. (B5) Lastly, peptides are classified in three categories based on their RM scores and the extent of modification is visualized: (i) RM score <0.5: peptide is likely differentially modified (magenta bars), (ii) 0.5 ≤ RM score<0.6: peptide is possibly differentially modified (blue bars), and (iii) RM score ≥0.6: peptide is likely not differentially modified (green bars).