Fig 2: Global PARylation stoichiometry is low but spans large dynamic range.A PARylated proteins were quantitatively adsorbed from crude cell lysates using BAB. Cell lysates (shPARG HCT116 cells treated with 2 mM H2O2) were incubated with BAB twice. The extent of PARylation in the original sample and in two flow-through fractions was assessed using immunoblotting assays with an anti-PAR antibody. B Flowchart illustrating the overall strategy for determining global PARylation stoichiometries. Light shPARG HCT116 cells were treated with H2O2 (2 mM, 5 min) to induce protein PARylation, while heavy cells were treated with Olaparib (1 μM, 40 min) to suppress basal PARylation levels. Enriched PARylated proteins from light cells were subject to on-beads digestion. Light and heavy peptides were then mixed at different protein ratios in the whole cell lysates. C Biological replicate experiments were conducted using SILAC HCT116 shPARG cells to determine PARylation stoichiometry. Set 1 identified 215 PARylated proteins, while Set 2 identified 226 PARylated proteins. D Reproducibility of PARylation stoichiometry between biological replicates. E Overall distribution of protein PARylation stoichiometries (n = 235). F Distribution of PARylation stoichiometry for grouped PARylated proteins (n = 235). G PARylation stoichiometry of previously well-studied PARP1 substrates. H Dynamic profiles of the median ratio between heavy and light at various SILAC cell lysate mix ratios for individual proteins were compared between biological replicates. I Validation of PARylation stoichiometry for PCNA and EWSR1. HCT116 shPARG cells treated with H2O2 (2 mM, 5 min) were lysed. Lysates were subject to boronate beads pull-down, and the PARylated proteins were eluted as described before. The eluted eluates and the serially diluted cell lysates input were analyzed by immunoblotting to validate PARylation stoichiometry.

Fig 3: Global PARylation stoichiometry is low but spans a large dynamic range. (A) PARylated proteins were quantitatively adsorbed from crude cell lysates using BAB. Cell lysates (shPARG HCT116 cells treated with 2 mM H2O2) were incubated with BAB twice. The extent of PARylation in the original sample and in two flow-through fractions was assessed using immunoblotting assays with an anti-PAR antibody. (B) Flowchart illustrating the overall strategy for determining global PARylation stoichiometries. Light shPARG HCT116 cells were treated with H2O2 (2 mM, 5 min) to induce protein PARylation, while heavy cells were treated with Olaparib (1 μM, 40 min) to suppress basal PARylation levels. Enriched PARylated proteins from light cells were subject to on-beads digestion. Light and heavy peptides were then mixed at different protein ratios in the whole-cell lysates. (C) Biological replicate experiments were conducted using SILAC HCT116 shPARG cells to determine PARylation stoichiometry. Set 1 identified 215 PARylated proteins, while Set 2 identified 226 PARylated proteins. (D) Reproducibility of PARylation stoichiometry between biological replicates. (E) Overall distribution of protein PARylation stoichiometries (n = 235). (F) Distribution of PARylation stoichiometry for grouped PARylated proteins (n = 235). (G) PARylation stoichiometry of previously well-studied PARP1 substrates. (H) Dynamic profiles of the median ratio between heavy and light at various SILAC cell lysate mix ratios for individual proteins were compared between biological replicates. (I) Validation of PARylation stoichiometry for PCNA and EWSR1. HCT116 shPARG cells treated with H2O2 (2 mM, 5 min) were lysed. Lysates were subject to boronate beads pull-down, and the PARylated proteins were eluted as described before. The eluted eluates and the serially diluted cell lysate input were analyzed by immunoblotting to validate PARylation stoichiometry. (J) Validation of PARylation stoichiometry for PCNA and TAF15 in wild-type cells. Wild-type HCT116 cells treated with H2O2 (2 mM, 5 min) and/or Olaparib (1 μM, 40 min) were lysed. Lysates were subject to boronate beads pull-down, and the PARylated proteins were eluted as described before. Input lysates (Total) and boronate-enriched pull-down fractions (PARylated proteins) were analyzed by immunoblotting with antibodies against PCNA, TAF15, and PAR to validate PARylation stoichiometry.