Figure 2: Quantitative identification of sumoylated residues in wild-type and slx8-1 and ufd1ΔCt^213–342 mutants.

(a) Two-step enrichment for sumoylated peptides using His₆SUMO^L109K strains. (b) Experimental design for SILAC experiments (see text for details). (c) Overview of peptides quantified in the triplicate SILAC experiments. Only diGly–Lys peptides for which SILAC ratios were obtained in at least two biological replicates were used in the analyses. In total, 171 distinct diGly–Lys peptides were quantified in both the slx8-1/ wild-type and slx8-1/ufd1ΔCt^213–342 IPs, which enabled us to calculate ufd1ΔCt^213–342/ wild-type ratios. See also Supplementary Fig. 2. (d) The normalized log₂ ratios of quantified diGly–Lys-modified peptides for ufd1ΔCt^213–342/ wild type and for slx8-1/wild type were plotted on a tsMAP, giving an overview of the relative change in abundance of individual peptides in each mutant. The two data sets correlate with a Pearson coefficient of r=0.49. Coloured data points represent peptides originating from the same proteins or from functionally related proteins, identified in Fig. 5. (e,f) Changes in protein sumoylation are not generally correlated with changes in protein concentration in either the slx8-1 or ufd1ΔCt^213–342 mutant. Among the proteins with quantified sumoylation sites, 112 and 79 were also quantified in the total proteome analyses of crude lysates for slx8-1/wild type and ufd1ΔCt^213–342/wild type, respectively. Log₂ SILAC ratios for sumoylated peptides were plotted against quantified ratios obtained for these proteins, yielding the maps in e and f.