Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography–Tandem Mass Spectrometry
ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography–high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.
Key wordsADP-ribosylation ADP-ribosylome Mass spectrometry Proteomics Af1521 macrodomain enrichment Affinity purification PARG
Ms. Monika Fey is acknowledged for the expression and purification of recombinant human PARG (University of Zurich) and Paolo Nanni for technical support for the MS measurements (FGCZ, University of Zurich). We also thank Felix R. Althaus (Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse) for providing hPARG expressing baculo virus. Stephan Christen and Deena Leslie Petrioli provided editorial assistance and critical input during the writing (University of Zurich). The work carried out in the laboratory of MLN was in part supported by the Novo Nordisk Foundation Center for Protein Research; the Novo Nordisk Foundation (grant number NNF14CC0001 and NNF13OC0006477); the Lundbeck Foundation (Grant number R171-2014-1496); The Danish Council of Independent Research, grant agreement number DFF 4002-00051 (Sapere Aude) and grant agreement number DFF 4183-00322A. ADP-ribosylation research in the laboratory of MOH is funded by the Kanton of Zurich, the University Research Priority Program (URPP) in Translational Cancer Biology at the University of Zurich, and the Swiss National Science Foundation (grant 310030B_138667).
- 1.Haigis MC, Mostoslavsky R, Haigis KM, Fahie K, Christodoulou DC, Murphy AJ, Valenzuela DM, Yancopoulos GD, Karow M, Blander G, Wolberger C, Prolla TA, Weindruch R, Alt FW, Guarente L (2006) SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126(5):941–954. doi: 10.1016/j.cell.2006.06.057 CrossRefPubMedGoogle Scholar
- 2.Rack JG, Morra R, Barkauskaite E, Kraehenbuehl R, Ariza A, Qu Y, Ortmayer M, Leidecker O, Cameron DR, Matic I, Peleg AY, Leys D, Traven A, Ahel I (2015) Identification of a class of protein ADP-ribosylating sirtuins in microbial pathogens. Mol Cell 59(2):309–320. doi: 10.1016/j.molcel.2015.06.013 CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Zhang Y, Wang J, Ding M, Yu Y (2013) Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome. Nat Methods 10(10):981–984. doi: 10.1038/nmeth.2603. http://www.nature.com/nmeth/journal/v10/n10/abs/nmeth.2603.html#supplementary-information CrossRefPubMedGoogle Scholar
- 15.Dani N, Stilla A, Marchegiani A, Tamburro A, Till S, Ladurner AG, Corda D, Di Girolamo M (2009) Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome. Proc Natl Acad Sci U S A 106(11):4243–4248. doi: 10.1073/pnas.0900066106. 0900066106 [pii]CrossRefPubMedPubMedCentralGoogle Scholar