Figure 6: PRISM combined with mass spectrometry reveals 751 unique SUMOylation sites.

(a) A schematic overview of PRISM adapted to system-wide proteomics. 1. Cells are lysed under denaturing conditions, completely inactivating endogenous proteases. 2. SUMOylated proteins are pre-enriched using, in this case, Ni-NTA pulldown to capture the histidine tag. 3. SUMOylated proteins are acetylated on-beads using SNHSA under highly denaturing conditions, enabling efficient blocking of lysines. 4. Following elution, proteins are concentrated over a 100 kDa filter under denaturing conditions, specifically removing free SUMO but retaining SUMO-modified proteins. 5. Concentrated lysine-blocked SUMOylated proteins are treated with SENP2, efficiently removing SUMO-2 and freeing up the lysines SUMO-2 was conjugated to. 6. DeSUMOylated proteins are concentrated over a 100 kDa filter under denaturing conditions, removing SUMO cleaved off by SENP2 while retaining proteins that were previously SUMO modified. 7. Concentrated SUMO target proteins are trypsinized, with trypsin only able to cleave arginines and lysines that were freed by SENP2. Thus, two reporter peptides are generated per site, either ending in a lysine or preceded by a lysine. 8. Peptides are analysed using high-resolution mass spectrometry. (b) SUMOylated proteins were purified from medium and heavy SILAC-labelled HeLa cells stably expressing His-tagged SUMO-2 (PD: His), using PRISM as described in a. During various steps of the purification, samples were taken for diagnostic purposes. Samples were size-separated using SDS–PAGE, transferred to membranes, probed using a SUMO-2/3 antibody and visualized using chemiluminescence. Samples are indicated by the corresponding step number from a. The experiment was performed in SILAC label-swapped biological duplicate. (c) An overview of all identified peptides, putative SUMO target proteins, SUMO target proteins dynamic in response to heat shock and all reporter peptides mapping to unique SUMOylation sites. A small number of sites was identified by multiple reporter peptides. (d) A schematic overview of the Andromeda confidence scores for all peptides mapping to SUMOylation sites. (e) Scatter plot analysis depicting correlation between the SILAC log₂ ratios of all SUMO sites identified in the label-swapped heat shock experiment. Blue dots represent upregulated SUMO sites, and red dots represent downregulated SUMO sites. Pearson correlation is indicated.