Proteomic analysis of SUMOylation in the post-ischemic brain
Post-translational protein modification by small ubiquitin-like modifier (SUMO) is increasingly recognized as a key modulator in many cellular processes including DNA repair, cell-cycle regulation, gene transcription, RNA processing, and protein quality control. This modification (SUMOylation) has been implicated in a variety of human diseases of major clinical significance. For example, brain ischemia/reperfusion dramatically activates global protein SUMOylation, which is believed to protect the brain from ischemic injury. Thus, identifying the proteins that are SUMOylated in the post-ischemic brain will provide insight into this endogenous neuroprotective response and may inform the development of new therapeutic strategies. Recent advancement in SUMO proteomics has established reliable methods for systematic characterization of SUMO targets in cells. However, identification of disease-related SUMOylated proteins in complex tissue samples is still technically challenging. Here, we provide a detailed protocol that uses SUMO transgenic mice to characterize SUMOylated proteins in post-ischemic brain samples. We describe the approach and procedures for nuclear fraction preparation, affinity purification of SUMOylated proteins, mass spectrometric data collection, data analysis, and verification of bona fide SUMO targets. This method may be adapted for any tissue sample for analysis of the SUMOylated proteome related to diseases that can be modeled in SUMO transgenic mice.
