Figure 4. The degradation capacity of oxidized proteasome is reduced compared to the naïve proteasome.

(A) Volcano plot shows peptides digested significantly different when an oxidized proteasome (P*) is used instead of the naïve proteasome (P) for digestion of naïve lysate (L, left) and oxidized lysate (L*, right). Each point represents a peptide measured. The color indicates significant change (red, abs (log2 (FC)) > 1 & adj. P value < 0.01). Increased FC indicates that the peptide is digested more when naïve proteasome is used. P values were calculated using two-sided t test, followed by Benjamini–Hochberg correction, n = 4 replicates. (B) Validation of the purity and activity of the naïve and oxidized 20S proteasome complexes that were added to the lysate. The purity of the complexes was assessed by SDS-PAGE analysis followed by coomassie staining, and the activity of the 20S proteasomes was analyzed by incubating proteasomes with the MV151 activity-based probe before separation by SDS-PAGE. (C) Quantification of time-dependent degradation assays of α-synuclein (αsyn) in the presence of naïve (P) and oxidized (P*) 20S proteasomes. The graph represents the averages of three independent experiments with SD. (D) PiP-MS results of α-synuclein degradation in different conditions. Values were calculated from a single experiment with n = 4 replicates for each condition. (E) Average disorder of proteins that are not degraded (Not deg-.), are degraded only in control lysate (L), only in oxidized lysate (L*) or in both lysates (L&L*). Values were calculated from a single experiment with n = 4 replicates for each condition. Significance is determined using two-sided Wilcoxon test (ns = P value > 0.05, ****P value < 0.0001). Horizontal lines define the median and boxes the 25th and 75th percentiles; whiskers represent the maximum and minimum values. Source data are available online for this figure.