Figure 1.

Establishing a peptide Arg-GlcNAc enrichment approach to identify endogenously modified targets. A, stable expression of FLAG-NleB1 and FLAG-NleB1^DXD for 24 h results in arginine GlcNAcylation of multiple host proteins only within NleB1. B, label-free quantification of Arg-GlcNAc peptide pulldown results from induction of NleB1 versus NleB1^DXD. The scatter plot shows the mean ion intensity peptide ratios of NleB1 versus NleB1^DXD plotted against negative logarithmic t test p values from biological triplicate experiments. Within biological replicates of NleB1-expressing cells, multiple putative arginine-glycosylated proteins were identified with manually curated Arg-GlcNAc peptides shown in red and Arg-GlcNAc peptides containing previously known Arg-GlcNAc modifications shown in purple. C and D, manually curated MS-MS spectra showing the previously known Arg-GlcNAc site of FADD Arg¹¹⁷ and the novel site Arg¹⁶ within charged multivesicular body protein 2a. HexNAc, N-acetylhexosamine.