Figure 2. Analysis of the mass spectrometry data reveals highly enriched interaction clusters.

A) Overview of proteins identified by the SILAC protein-level workflow, by the label-free sites-level workflow, and proteins in which sites were identified. Only proteins which were found by both approaches, shaded in blue, were used for further analysis.

B) Scatter plot analysis of protein SUMOylation ratio (log₂) in response to MMS, versus protein SUMOylation SILAC ratio (log₂) enrichment over the parental control. The parental control refers to cells not expressing FLAG-SUMO-2. “Dynamic” proteins were found consistently and significantly changed across all proteomic analyses and replicates. Proteins of interest which were confirmed by immunoblotting analysis are indicated.

C) Volcano plot displaying SUMOylation site ratio (log₂) in response to MMS, versus the negative P value (log₁₀) resulting from permutation-based FDR. Red dots indicate downregulated sites, blue dots indicate upregulated sites. Sites in proteins of interest are indicated.

D) Term enrichment analysis of identified up- and down-regulated SUMOylated proteins in response to MMS. Relative score of regulated SUMOylated proteins versus the human proteome is indicated for the Gene Ontology (GO) Molecular Functions, Cellular Compartments and Biological Processes categories. A full term enrichment analysis is available for upregulated SUMO targets (Table S8) and downregulated SUMO targets (Table S9).

E) STRING network analysis of all regulated SUMOylated proteins in response to MMS. Enrichment ratio refers to the amount of interactions observed versus the amount of expected interactions (p>0.4).