Fig. 2: Side-by-side comparison of DDA and DIA for ubiquitinomics.

a Schematic of data-dependent acquisition (DDA) and data-independent acquisition (DIA) strategies. A K-GG peptide sample was prepared from MG-132-treated HCT116 cells and four replicates were acquired with each scan mode. The DDA data were processed with MaxQuant (MBR enabled). For DIA data processing in DIA-NN, the same DIA-MS raw files were processed with either library-free (no experimental spectral library) or library-based search (experimental spectral library obtained through high pH reversed-phase fractionation of peptides prior to K-GG peptide enrichment). b Quantified K-GG peptides with 2 mg of total protein as input for each replicate, in a side-by-side comparison of DDA and DIA-MS. Four samples (injection replicates) were acquired with 75 min single-shot LC-MS runs and processed with MaxQuant or DIA-NN. Library-based DIA data analysis was performed with an ultra-deep K-GG peptide library generated by library-free DIA analysis of high-pH fractions, as detailed in “Methods” (LF = library-free, lib = experimentally-generated spectral library). c Coefficients of variation (CVs, in %) for K-GG peptides as shown in b are plotted for each method. Continuous lines demarcate the median and dashed lines the upper and lower quartiles. d Number of identifications with 0, 1, 2, or 3 missing values in four replicates (samples shown in b) for DDA and DIA. e Venn diagram showing overlap of ubiquitinated peptides (K-GG) identified by DDA and by DIA (DIA-NN’s library-free (LF) and spectral library-based (lib) approaches). f Scatter plot of Log2-transformed K-GG peptide intensities obtained from library-free and spectral library-based search options in DIA-NN (n = 4, injection replicates). Values indicate Pearson correlation coefficients. g Raw data of MG-132-treated HEK293 cells from Hansen et al. (Fig. 1d in the original manuscript, three workflow replicates, each injected in duplicate and processed with Spectronaut’s directDIA mode¹⁶) were processed with DIA-NN (library-free mode). Plots show identified K-GG peptides (unique peptide sequences) for both Software packages. Source data are provided as a Source Data file.