We used RutR and specific biotin-DNA to validate the efficiency of SPIDER capturing protein-nucleic acid interaction. Pupylation sites on RutR after SPIDER assay was identified by LC-MS/MS analysis, by searching for an additional mass of ~243 Da, which represents the three C-terminal residues of Pup, i. e., GGE, that covalently binds to adjacent lysine by pupylation. ）