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In this study, we improved the most commonly used methods for MS detection of SUMOylated sites and used an E. coli recombination SUMOylation system with SUMO-1 (T95R). This system provides fast enrichment of SUMOylated viral protein in less than 2 days, and shows advantage over the method of collecting modified protein from cells in convenience and sensitivity. Furthermore, this method provides an option for rapid and accurate identification of the potential viral protein SUMOylation sites.