Fig. 1: Comparison of urea- and SDC-based cell lysis for mass spectrometry-based ubiquitinomics.

a Fraction of unmodified and K-GG modified peptides quantified from either urea or SDC lysates in MG-132-treated HCT116 cells. Four individual samples were processed for each lysis protocol, with 2 mg of protein input per replicate. Only half of each sample was injected into the MS, which was operated in DDA mode (125 min LC gradient). The raw data were processed with MaxQuant, with “match between runs” (MBR) enabled. Gray dots show cumulative numbers of K-GG peptide identifications from four replicates. b Overlap of quantified K-GG peptides and K-GG modified proteins with urea and SDC lysis buffers. c MS-quantified ubiquitinated peptides (K-GG remnants) with different protein inputs in MG-132-treated Jurkat cells (6 h). Four individual samples were processed for each condition and the data were acquired in single-shot mode using a 125 min DDA-MS method. The raw files were processed with MaxQuant, with match-between-runs (MBR) activated. d Fraction of unmodified and K-GG modified peptides quantified with the SDC-based lysis protocol and with UbiSite¹⁹. Three single-shot runs of enriched K-GG peptides from MG-132-treated Jurkat cells (2 mg of input, three replicates of samples as shown in c) were processed with MaxQuant (with MBR) together with three biological replicates of bortezomib-treated Jurkat samples (16 high pH-reversed phase fractions for each replicate) from Akimov et al. e Ranked K-GG peptide coefficients of variation (CVs) for samples shown in d. The 20% CV cut-off is marked. Source data are provided as a Source Data file.