Fig. 3: DIA-MS ubiquitinomics for untreated samples and for high-throughput applications.

a HCT116 cells were treated with DMSO or 10 µM MG-132 for 6 h and K-GG peptides from different protein inputs quantified by 75 min single-run DIA-MS (library-free mode). Four samples were processed independently for each tested protein input. b Dot plot of Log2-transformed K-GG peptide ratios (MG-132 vs DMSO) with 2 mg of protein input, as shown in a. Selected K-GG sites are highlighted. c Number of quantified K-GG peptides with 15, 30, 45, 60, and 75 min DIA-MS runs. A K-GG peptide pool was injected on the same instrument for each method (n = 4, injection replicates). The data were processed with library-free DIA. d Coefficients of variation (CVs) distributions for K-GG peptides quantified in c. Continuous lines in the violin plots demarcate the median and dashed lines upper and lower quartiles. e Shown are K-GG peptide numbers from DMSO-treated U2OS cells processed by Hansen et al. (Fig. 3 in the original manuscript, three biological replicates, each injected twice¹⁶) and the same raw files processed with DIA-NN (library-free mode). Source data are provided as a Source Data file.