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).