Skip to main content
SpringerLink
Log in

Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry

  • Protocol
  • First Online:
Nuclear Bodies and Noncoding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1262))

Abstract

Determining the proteome of a nuclear body is a crucial step toward understanding its function; however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-based affinity purification followed by quantitative mass spectrometry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 39.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 54.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 54.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mao YS, Zhang B, Spector DL (2011) Biogenesis and function of nuclear bodies. Trends Genet 27:295–306. doi:10.1016/j.tig.2011.05.006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Andersen JS, Lyon CE, Fox AH et al (2002) Directed proteomic analysis of the human nucleolus. Curr Biol 12:1–11. doi:10.1016/S0960-9822(01)00650-9

    Article  PubMed  Google Scholar 

  3. Andersen JS, Lam YW, Leung AKL et al (2005) Nucleolar proteome dynamics. Nature 433:77–83. doi:10.1038/nature03207

    Article  CAS  PubMed  Google Scholar 

  4. Boisvert F-M, Lamond AI (2010) p53-Dependent subcellular proteome localization following DNA damage. Proteomics 10:4087–4097. doi:10.1002/pmic.201000213

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Scherl A, Couté Y, Déon C et al (2002) Functional proteomic analysis of human nucleolus. Mol Biol Cell 13:4100–4109. doi:10.1091/mbc.E02-05-0271

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Saitoh N et al (2004) Proteomic analysis of interchromatin granule clusters. Mol Biol Cell 15:3876–3890. doi:10.1091/mbc.E04-03-0253

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Lam YW, Lyon CE, Lamond AI (2002) Large-scale isolation of Cajal bodies from HeLa cells. Mol Biol Cell 13:2461–2473. doi:10.1091/mbc.02-03-0034

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Mosley AL, Florens L, Wen Z, Washburn MP (2009) A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus. J Proteomics 72:110–120. doi:10.1016/j.jprot.2008.10.008

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Franklin S, Zhang MJ, Chen H et al (2011) Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy. Mol Cell Proteomics 10:M110.000703. doi:10.1074/mcp.M110.000703

    Article  PubMed Central  PubMed  Google Scholar 

  10. Foster LJ, de Hoog CL, Zhang Y et al (2006) A mammalian organelle map by protein correlation profiling. Cell 125:187–199. doi:10.1016/j.cell.2006.03.022

    Article  CAS  PubMed  Google Scholar 

  11. Van Damme E, Laukens K, Dang TH, Van Ostade X (2010) A manually curated network of the PML nuclear body interactome reveals an important role for PML-NBs in SUMOylation dynamics. Int J Biol Sci 6:51–67. doi:10.7150/ijbs.6.51

    Article  PubMed Central  PubMed  Google Scholar 

  12. Bond CS, Fox AH (2009) Paraspeckles: nuclear bodies built on long noncoding RNA. J Cell Biol 186:637–644. doi:10.1083/jcb.200906113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Sutherland HGE et al (2001) Large-scale identification of mammalian proteins localized to nuclear sub-compartments. Hum Mol Genet 10:1995–2011. doi:10.1093/hmg/10.18.1995

    Article  CAS  PubMed  Google Scholar 

  14. Fong K-W, Li Y, Wang W et al (2013) Whole-genome screening identifies proteins localized to distinct nuclear bodies. J Cell Biol 203:149–164. doi:10.1083/jcb/201303145

    Article  PubMed Central  PubMed  Google Scholar 

  15. Naganuma T, Nakagawa S, Tanigawa A et al (2012) Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J 31:4020–4034. doi:10.1038/emboj.2012.251

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Schulze WX, Mann M (2004) A novel proteomic screen for peptide-protein interactions. J Biol Chem 279:10756–10764. doi:10.1074/jbc.M309909200

    Article  CAS  PubMed  Google Scholar 

  17. Butter F, Scheibe M, Mörl M, Mann M (2009) Unbiased RNA-protein interaction screen by quantitative proteomics. Proc Natl Acad Sci U S A 106:10626–10631. doi:10.1073/pnas.0812099106

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Tsai BP, Wang X, Huang L, Waterman ML (2011) Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach. Mol Cell Proteomics 10:M110.007385–M110.007385. doi:10.1074/mcp.M110.007385

    Article  PubMed Central  PubMed  Google Scholar 

  19. Ong S-E, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  CAS  PubMed  Google Scholar 

  20. Trinkle-Mulcahy L, Boulon S, Lam YW et al (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 183:223–239. doi:10.1083/jcb.200805092

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Trinkle-Mulcahy L (2012) Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry. Proteomics 12:1623. doi:10.1002/pmic.201100438

    Article  CAS  PubMed  Google Scholar 

  22. Trinkle-Mulcahy L, Andersen J, Lam YW et al (2006) Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability. J Cell Biol 172:679–692. doi:10.1083/jcb.200508154

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Chamousset D, Mamane S, Boisvert F-M, Trinkle-Mulcahy L (2010) Efficient extraction of nucleolar proteins for interactome analyses. Proteomics 10:3045–3050. doi:10.1002/pmic.201000162

    Article  CAS  PubMed  Google Scholar 

  24. Chamousset D, De Wever V, Moorhead GB et al (2010) RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits. Mol Biol Cell 21:4212–4226. doi:10.1091/mbc.E10-04-0287

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Selbach M, Mann M (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Method 3:981–983. doi:10.1038/nmeth972

    Article  CAS  Google Scholar 

  26. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224. doi:10.1126/science.1124618

    Article  CAS  PubMed  Google Scholar 

  27. Cox J, Matic I, Hilger M et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4:698–705. doi:10.1038/nprot.2009.36

    Article  CAS  PubMed  Google Scholar 

  28. Wang B, Malik R, Nigg EA, Körner R (2008) Evaluation of the low-specificity protease elastase for large-scale phosphoproteome analysis. Anal Chem 80:9526–9533. doi:10.1021/ac801708p

    Article  CAS  PubMed  Google Scholar 

  29. Boulon S, Ahmad Y, Trinkle-Mulcahy L et al (2010) Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics 9:861–879. doi:10.1074/mcp.M900517-MCP200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Rothbauer U, Zolghadr K, Muyldermans S et al (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289. doi:10.1074/mcp.M700342-MCP200

    Article  CAS  PubMed  Google Scholar 

  31. Wang X, Huang L (2008) Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol Cell Proteomics 7:46–57. doi:10.1074/mcp.M700261-MCP200

    Article  PubMed  Google Scholar 

  32. Mousson F, Kolkman A, Pijnappel WWMP et al (2008) Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes. Mol Cell Proteomics 7:845–852. doi:10.1074/mcp.M700306-MCP200

    Article  CAS  PubMed  Google Scholar 

  33. Fang L, Wang X, Yamoah K et al (2008) Characterization of the human COP9 signalosome complex using affinity purification and mass spectrometry. J Proteome Res 7:4914–4925. doi:10.1021/pr800574c

Download references

Acknowledgments

The authors would like to thank Drs. Angus Lamond, Nick Morrice, Douglas Lamont, and Lawrence Puente for advice and assistance. We thank colleagues in the Fox and Trinkle labs for helpful discussions and suggestions and Anna Kula and Dr. Alessandro Marcello for assistance with the alternate nuclear isolation protocol. This work was supported by the Terry Fox Research Institute (Ref: 20148, LTM), Natural Sciences and Engineering Research Council (Ref: 372370, LTM), and National Institute of Health and Medical Research, Australia (Ref: 1030695, 1048659, and 1050585, AHF). LTM holds a Canadian Institutes of Health Research New Investigator Award. AHF is a Cancer Council of Western Australia Research Fellow.

Author information

Authors and Affiliations

  1. Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, the University of Western Australia, Crawley, Western Australia, 6009, Australia

    Archa Fox

  2. Centre de Recherche de Biochimie Macromoléculaire UMR 5237 CNRS, Université Montpellier, Montpellier, France

    Archa Fox

  3. Department of Cellular and Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5

    Virja Mehta

  4. Department of Cellular and Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5

    Laura Trinkle-Mulcahy

  5. Centre de Recherche de Biochimie Macromoléculaire UMR 5237 CNRS, Université Montpellier, Montpellier, France

    Severine Boulon

Authors
  1. Archa Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Virja Mehta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Severine Boulon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laura Trinkle-Mulcahy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Archa Fox or Laura Trinkle-Mulcahy .

Editor information

Editors and Affiliations

  1. RNA Biology Laboratory, RIKEN, Saitama, Japan

    Shinichi Nakagawa

  2. Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

    Tetsuro Hirose

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Fox, A., Mehta, V., Boulon, S., Trinkle-Mulcahy, L. (2015). Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry. In: Nakagawa, S., Hirose, T. (eds) Nuclear Bodies and Noncoding RNAs. Methods in Molecular Biology, vol 1262. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2253-6_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2253-6_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2252-9

  • Online ISBN: 978-1-4939-2253-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation