Supplementary Figure 1: Enrichment of ubiquitinated peptides by UbiSite.
From: UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites
a, Sequence alignment of remnants after LysC digestion of NEDD8, ubiquitin and ISG-15. b, Comparison of UbiSite and VU1 antibodies in recognizing ubiquitinated species in Hep2 cells treated with vehicle (DMSO) or proteasomal inhibitor (5 μM, 8 h) by western blotting. Shown is a representative of three independent experiments. c, Overlap between unique ubiquitination sites in samples digested with LysC only or trypsin plus LysC. d, Proportions of identified ubiquitinated and non-ubiquitinated peptides in samples treated with LysC only or LysC followed by trypsin. e, Proportions of the summed peptide intensities in samples treated with LysC only or LysC followed by trypsin. f, Elution profiles of ESTLHLVLR and K48 ubiquitin chain peptides over all offline high-pH (HpH) fractions summarized for the three replicates of Hep2 cells treated with bortezomib. The bottom panel shows the number of identified ubiquitinated peptides within each fraction and replicate for the same cells and treatment. g, Drop in the number of peptide identifications during elution of the highly abundant ESTLHLVLR and K48 ubiquitin chain peptides. Top panels show the total ion chromatograms for HpH fractions 5 and 12, as indicated. Red dotted lines provide the number of peptide-to-spectrum matches (PSMs) throughout the LC gradient, binned in 5-min windows. The bottom panel shows the total MS1 spectrum for fraction 12 during the 20-min elution of the two abundant peptides.
