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Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome

Nature Methods volume 10, pages 981984 (2013) | Download Citation

Abstract

Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid– and glutamic acid–ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.

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Change history

  • Corrected online 26 August 2013

    In the version of this article initially published online, the x-axis tick label "≥15" in Figure 3e was mislabeled. The error has been corrected for the print, PDF and HTML versions of this article.

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References

  1. 1.

    , & Biochem. Biophys. Res. Commun. 11, 39–43 (1963).

  2. 2.

    , , , & Nat. Rev. Cancer 10, 293–301 (2010).

  3. 3.

    & Mol. Cell 39, 8–24 (2010).

  4. 4.

    et al. Nat. Biotechnol. 30, 283–288 (2012).

  5. 5.

    et al. Nature 434, 917–921 (2005).

  6. 6.

    Nat. Biotechnol. 29, 1078–1079 (2011).

  7. 7.

    et al. Proc. Natl. Acad. Sci. USA 106, 4243–4248 (2009).

  8. 8.

    & Int. J. Mass Spectrom. 312, 114–121 (2012).

  9. 9.

    , , & Microbiol. Mol. Biol. Rev. 70, 789–829 (2006).

  10. 10.

    , & J. Am. Chem. Soc. 131, 14258–14260 (2009).

  11. 11.

    & Methods Mol. Biol. 147, 119–128 (2000).

  12. 12.

    , & J. Biol. Chem. 258, 6466–6470 (1983).

  13. 13.

    , , & J. Proteome Res. 12, 1868–1880 (2013).

  14. 14.

    et al. Nat. Struct. Mol. Biol. 19, 685–692 (2012).

  15. 15.

    et al. Science 332, 1322–1326 (2011).

  16. 16.

    et al. Nat. Cell Biol. 13, 623–629 (2011).

  17. 17.

    et al. Clin. Cancer Res. 18, 510–523 (2012).

  18. 18.

    , , , & Clin. Cancer Res. 18, 1655–1662 (2012).

  19. 19.

    , , & Science 336, 728–732 (2012).

  20. 20.

    & Biochem. Biophys. Res. Commun. 285, 27–31 (2001).

  21. 21.

    et al. Mol. Cell. Proteomics 8, 2759–2769 (2009).

  22. 22.

    et al. Cell 143, 1174–1189 (2010).

  23. 23.

    et al. Mol. Cell 44, 325–340 (2011).

  24. 24.

    & Nat. Biotechnol. 23, 1391–1398 (2005).

  25. 25.

    , & J. Mol. Biol. 220, 507–530 (1991).

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Acknowledgements

We thank S. McKnight and H. Yu for discussions. We thank H. Yu, H. Mirzaei and S. Gygi for access to the tissue culture, mass spectrometry and computation facilities, respectively. pCMV-Sport6-GAR1 and pCMV-Sport6-DDX21 were kind gifts from L. Lum (University of Texas Southwestern Medical Center). This work was supported by grants from the UT Southwestern Medical Center Endowed Scholar Program, the Cancer Prevention and Research Institute of Texas (CPRIT R1103) and the Welch Foundation (I-1800) to Y.Y. Y.Y. is a Virginia Murchison Linthicum Scholar in Medical Research and a CPRIT Scholar in Cancer Research.

Author information

Affiliations

  1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Yajie Zhang
    • , Jianqi Wang
    • , Ming Ding
    •  & Yonghao Yu

Authors

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Contributions

Y.Y. conceived the project and designed the approach. Y.Y., Y.Z. and M.D. performed research and analyzed data. Y.Y. and J.W. performed computational analyses. Y.Y. supervised the project. Y.Y. wrote the manuscript with input from all coauthors.

Competing interests

A US patent on the method described in this manuscript, along with the sites identified, has been filed (no. 13/874,453 to Y.Y.).

Corresponding author

Correspondence to Yonghao Yu.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results and Supplementary Figures 1–6

Excel files

  1. 1.

    Supplementary Table 1

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shPARG HCT116 cells using 0.2 mM H2O2 stimulation. Description of the column headings is included. ## indicates the reverse hits.

  2. 2.

    Supplementary Table 2

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shPARG HCT116 cells using 2 mM H2O2 stimulation.

  3. 3.

    Supplementary Table 3

    Asp- and Glu-ADP-ribosylated peptides identified and quantified (as shown in Fig. 3a) using single Lys labeling (Lys0 and Lys8 for light and heavy, respectively). Data from the two technical replicate analyses were included. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with olaparib, then stimulated with 2 mM H2O2. Additional information about the headings (for peptide quantitation) is included.

  4. 4.

    Supplementary Table 4

    Asp- and Glu-ADP-ribosylated peptides identified and quantified using double labeling of Lys and Arg (Lys0/Arg0 and Lys8/Arg10 for light and heavy, respectively). Data from two technical replicate analyses were included. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with olaparib, then stimulated with 2 mM H2O2.

  5. 5.

    Supplementary Table 5

    Asp- and Glu-ADP-ribosylated peptides identified and quantified in response to A966492 treatment as shown in Fig. 3d. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with 1 mM A966492, then stimulated with 2 mM H2O2.

  6. 6.

    Supplementary Table 6

    Asp- and Glu-ADP-ribosylated peptides identified and quantified in response to AG14361 treatment as shown in Fig. 3d. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with 1 μM AG14361, then stimulated with 2 mM H2O2.

  7. 7.

    Supplementary Table 7

    Asp- and Glu-ADP-ribosylated peptides identified and quantified in response to iniparib treatment as shown in Fig. 3d. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with 50 μM iniparib, then stimulated with 2 mM H2O2.

  8. 8.

    Supplementary Table 8

    Asp- and Glu-ADP-ribosylated peptides identified and quantified in response to 3-AB treatment as shown in Fig. 3d. Light cells were stimulated with 2 mM H2O2 whereas heavy cells were pretreated with 50 μM 3-AB, then stimulated with 2 mM H2O2.

  9. 9.

    Supplementary Table 9

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shPARG HEK293T cells, using 2 mM H2O2 stimulation.

  10. 10.

    Supplementary Table 10

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shGFP HCT116 cells, using 2 mM H2O2 stimulation.

  11. 11.

    Supplementary Table 11

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shGFP and shPARG HCT116 cells in response to the treatment of 50 μM MNNG.

  12. 12.

    Supplementary Table 12

    Asp- and Glu-ADP-ribosylated peptides and proteins identified in shGFP HCT116 cells with no treatment of DNA-damaging agents.

  13. 13.

    Supplementary Table 13

    The complete list of the identified ADP-ribosylated sites. Modified sites (Asp and Glu) are aligned, with their ModScore (the highest score) and the number of identification shown. Also included is a tab summarizing the results of quantitative analyses of the ADP-ribosylated peptides in response to PARP inhibitor treatment, including olaparib, A966942, AG14361, iniparib and 3-AB. Only peptides with their ADP-ribosylation site(s) confidently-localized (ModScore ≥ 13) were included. For peptides that were identified multiple times, the one with the highest summed (Light+Heavy) intensity is reported. SNR, signal-to-noise ratio.

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DOI

https://doi.org/10.1038/nmeth.2603