Profiling of the ADP‐Ribosylome in Living Cells

Abstract Post‐translational modification (PTM) with ADP‐ribose and poly(ADP‐ribose) using nicotinamide adenine dinucleotide (NAD+) as substrate is involved in the regulation of numerous cellular pathways in eukaryotes, notably the response to DNA damage caused by cellular stress. Nevertheless, due to intrinsic properties of NAD+ e.g., high polarity and associated poor cell passage, these PTMs are difficult to characterize in cells. Here, two new NAD+ derivatives are presented, which carry either a fluorophore or an affinity tag and, in combination with developed methods for mild cell delivery, allow studies in living human cells. We show that this approach allows not only the imaging of ADP‐ribosylation in living cells but also the proteome‐wide analysis of cellular adaptation by protein ADP‐ribosylation as a consequence of environmental changes such as H2O2‐induced oxidative stress or the effect of the approved anti‐cancer drug olaparib. Our results therefore pave the way for further functional and clinical studies of the ADP‐ribosylated proteome in living cells in health and disease.

of the manuscript) (complete dataset see excel sheet). The significance of the enrichment is revealed as a function of the p-value, which indicates whether a biological process is significantly higher than random expectations. Figure 9: GO-term analysis of ADP-ribosylated proteins after olaparib treatment Proteins significantly enriched in ANOVA analysis in trace 5 and 6 of Figure 5A of the manuscript were classified according to Biological Process, Cellular Component and Molecular Function (using DAVID). 2 The significance of the enrichment is revealed as a function of the p-value, which indicates whether a biological process is significantly higher than random expectations. Figure 11: Overlap of ADP-ribosylated target proteins with target proteins reported in the literature. Venn diagrams show overlap between target proteins identified in this study with all known ADP-ribosylated target proteins in literature, with target proteins enriched with ADP-ribose macrodomains or -antibodies (yellow) [3][4][5] with target proteins enriched by borate affinity (blue) 6 and with target proteins enriched with nucleoside/nucleotide derivatives (red). 7-11, Supplementary Figur 12: GO-term analysis of ADP-ribosylated proteins: Proteins significantly enriched in ANOVA analysis in trace 3 was classified according to Biological Process, Cellular Component and Molecular Function (using DAVID). The significance of the enrichment is revealed as a function of the p-value, which indicates whether a biological process is significantly higher than random expectations.

Synthesis Section
General All reactions were conducted using standard laboratory techniques. If necessary, due to oxygen or water sensitivity, reactions were performed under the exclusion of air or moisture. In that case dry solvents (VWR) were used. Otherwise, solvents were p.A., absolute or HPLC grade.
Commercially available chemicals and solvents were purchased from TCI Chemicals, Sigma-Aldrich/Merck, Acros Organics, Carbosynth, Carl Roth, VWR, BonTac and ABCR. If not denoted differently, they were used without further purification. For chromatography, solvents were p.A., absolute or HPLC grade, technical solvents were distilled before use. For the purification with the HPLC and ion exchange chromatography, water from a combined reverse osmosis/ultrapure water system (Milli-Q) was used. Medium pressure liquid chromatography (MPLC) was performed on a PrepChrom C-700 (Büchi) instrument using SVF D22 RP-18 columns (Götec Labortechnik) with a linear gradient of Milli-Q water/Acetonitrile or 50 mM TEAB/Acetonitrile. The solvents are specified in the experimental procedures and are stated as volume (v/v) ratios.
The gradient was adjusted to the respective separation problem and is specified in the experimental procedures. All compounds were obtained as their triethylammonium salts after purification via HPLC.
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Avance III 400 MHz or 600 MHz spectrometers at room temperature (r.t.). All samples were dissolved in deuterated solvents (D 2 O) and the spectra were calibrated to the internal standards of the deuterated solvents (D 2 O: δ( 1 H) 4.79 ppm) or using 1 H spectrum as absolute reference (for 13 C and 31 P in D 2 O).
Chemical shifts (δ) are given in parts per million (ppm) and coupling constants in Hertz (Hz). For signal assignment two dimensional spectra (HSQC, HMBC, COSY, 31 P-HMBC) were used. NMR spectra were evaluated with MestReNova 12.0.4 (MestReLab Research S.L.) and the data were
The crude product was concentrated under reduced pressure, dissolved in water and purified by RP-HPLC (C18 HTec, 50 mM TEAB buffer/acetonitrile). After lyophilisation, the product was obtained as its triethylammonium salt (62 %, by UV detection). -60/40). After lyophilisation, the product was obtained as their triethylammonium salt (by UV detection).

Biochemical Methods
Biochemical in vitro assays for the acceptance of modified NAD + analogs

Cell viability of transfected cells
For the cytotoxicity of modified NAD + analogs on HeLa cells, the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) was used according to the instructions provided by the kit manufacturer.   5 mL) for 5 s at 500 g and room temperature. This step was repeated twice and the elution fraction of each samples were combined, lyophilized and stored at -20 °C.
Samples for SDS PAGE analysis were prepared from each fraction.

PARG treatment for degradation of PAR
Elution fractions were re-suspended in PARG reaction buffer (KH 2 PO 4 , 5 mM, pH 7.5; KCl, 5 mM, β-mercaptoethanol, 0.9 mM) and PARG (10 nM) was added. The mixtures were incubated for 4 hours at 37 °C. Afterwards, the reaction was quenched by the addition of 6x loading dye, denaturation for 5 min at 95 °C and resolved by SDS PAGE analysis (12.5 % separation gels, 35 mA, 25 min). Colloidal Coomassie staining and imaging was performed as indicated above.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 20 partner repository with the dataset identifier PXD020549 (Username: reviewer40609@ebi.ac.uk; Password: xNEgVG6t).