Fig. 3: Integration of boronic acid enrichment with ETD-based MS.

a Overview of the experimental design. HeLa cells were cultured in quadruplicate (n = 4), mock-treated or H₂O₂-treated at 1 mM for 10 min, lysed, and trypsinized. ADPr-modified peptides were enriched using boronate-affinity beads, eluted using 0.15% trifluoroacetic acid to maintain the intact ADPr moiety, and analyzed using ETD-based high-resolution MS. b Comparison of all ADPr enrichment methods employed in this study, visualizing peptide purity based on either abundance or spectral counting. Data are presented as mean values  ± SD, n = 4 cell culture replicates. “BA”; boronate affinity, “AB1”; E6F6A antibody, “AB2”; D9P7Z antibody. c Pie-chart analysis showing the distribution of ADPr sites across different amino acid residue types, either based on the number of sites or abundance. d Term enrichment analysis visualizing similarity of ADPr target proteins identified in this experiment to several other ADPr proteomics studies^{10,14,16,29,31,36,42}, to several cellular functions associated with ADPr, or to a deep total proteome study⁸⁰. The relative score is based on the multiplication of logarithms derived from the enrichment ratio and the q-value. Terms were significant with q < 0.02, as determined through Fisher Exact Testing with Benjamini–Hochberg correction. e Scaled Venn diagram depicting the overlap between ADPr target proteins identified using either; boronate affinity enrichment and detection of intact ADPr using ETD-based fragmentation (this experiment), boronate affinity enrichment and detection of hydroxamic acid derivatives using collision-induced dissociation (CID) fragmentation^10,31, or Af1521 enrichment and detection of intact ADPr using ETD-based fragmentation^14,16. CID fragmentation is comparable to HCD fragmentation. “BA”; boronate affinity, “hydrox. acid”; hydroxamic acid derivatives. Source data are provided as a Source Data file.