Identification of Arg44 and Arg663 in HERC5 as the major ADP-riboxanation sites
To determine the ADP-riboxanation sites in HERC5 catalyzed by S. flexneri effector OspC1, we perfomed LC-MS/MS analysis. Briefly, HERC5-Flag with WT or MUT OspC1 were co-overexpressed in 293T cells and HERC5-Flag proteins were immunoprecipitated with anti-Flag agarose and then separated by SDS-PAGE (Figure 1e). The target band corresponding to HERC5-Flag were harvested and subjected to in-gel digestion with trypsin, followed by LC-MS/MS analysis in collision-induced dissociation (CID) mode. Our analysis indicated that HERC5 was ADP-riboxanated at Arg44 and Arg663 sites by S. flexneri effector OspC1 (Figure 2a, b; Supplementary Figure 1).
The presence of diagnostic ions with m/z of 136.06, 250.09, 348.05 and 428.04 which are adenine+, adenosine - H2O+, adenosine monophosphate+ (AMP+), adenosine diphosphate+ (ADP+) in MS/MS spectra allowed the detection of putative ADP-riboxanated peptides (Li et al., 2021; Peng et al., 2022). However, due to the mass discrimination in CID mode, we only detected the diagnostic marker ions at m/z 348.07 (AMP+) and 428.04 (ADP+) (Figure 2a). We re-performed the LC-MS/MS analysis in high energy collision dissociation (HCD) mode, and fortunately detected all of the diagnostic ions mentioned above (Figure 2b; Supplementary Figure 1). By quantitative analysis, we determined that about 45% of total HERC5 were ADP-riboxanated at Arg44 by S. flexneri effector OspC1 (Figure 2c).