Fig. 4: Identification of mono-Asp/Glu-ADPr on additional targets through ester bond preservation.

A Schematic overview of the experimental workflow for enrichment of mono-ADPr peptides. Arg-C Ultra at a high protease-to-protein ratio (1:2000) is used for protein digestion, followed by peptide purification and mono-ADPr enrichment by the AbD43647 IgG antibody. Subsequent mass-spectrometry employs both HCD- and ETD-type fragmentation methods for high-confidence localization of the modification site. This schematic was generated in Adobe Illustrator using some graphical elements adapted from Longarini et al.¹⁷ B Pie-chart overview of the localized Asp/Glu-ADPr sites (delta score > 40, localization probability ≥ 0.9) from mono-ADPr enrichment in HPF1KO cells and comparison with published studies (See also Supplementary Data 2). C Overview of the number of ADPr sites per protein. D Top 11 proteins identified in our large-scale mono-ADPr enrichment experiments ranked by the cumulative intensity of the identified ADPr sites. E Schematic depiction of all high-confidence (delta score > 40, localization probability ≥ 0.9) HMGA1b mono-Asp/Glu-ADPr sites identified in HPF1KO cells on a linear model of HMGA1b. These sites have not been previously identified in other large-scale proteomic studies. F ETD spectrum showing unambiguous localization of the HMGA1bE17ADPr modification site. G Schematic depiction of all high-confidence (delta score > 40, localization probability ≥ 0.9) HMGN2 mono-Asp/Glu-ADPr sites identified in HPF1KO cells on a linear model of HMGN2 (NBD Nucleosome Binding Domain, RD Regulatory Domain). These sites have not been previously identified in other large-scale proteomic studies. H ETD spectrum showing unambiguous localization of the HMGN2E20ADPr modification site. Data for (B–H) is from a total of three biological replicates.