Skip to main content
SpringerLink
Log in

Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

  • Protocol
  • First Online:
Quantitative Proteomics by Mass Spectrometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1410))

Abstract

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

Similar content being viewed by others

References

  1. Rigaut G et al (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10):1030–1032

    Article  CAS  PubMed  Google Scholar 

  2. Cristea IM et al (2005) Fluorescent proteins as proteomic probes. Mol Cell Proteomics 4(12):1933–1941

    Article  CAS  PubMed  Google Scholar 

  3. Sueda S, Tanaka H, Yamagishi M (2009) A biotin-based protein tagging system. Anal Biochem 393(2):189–195

    Article  CAS  PubMed  Google Scholar 

  4. Lambert JP et al (2015) Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. J Proteomics 118:81–94

    Article  CAS  PubMed  Google Scholar 

  5. Kaltenbrun E et al (2013) A Gro/TLE-NuRD corepressor complex facilitates Tbx20-dependent transcriptional repression. J Proteome Res 12(12):5395–5409

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Greco TM et al (2011) Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation. Mol Cell Proteomics 10(2):M110.004317

    Article  PubMed Central  PubMed  Google Scholar 

  7. Domanski M et al (2012) Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. Biotechniques 0(0):1–6

    PubMed Central  PubMed  Google Scholar 

  8. Joshi P et al (2013) The functional interactome landscape of the human histone deacetylase family. Mol Syst Biol 9:672

    Article  PubMed Central  PubMed  Google Scholar 

  9. Pflieger D et al (2008) Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation. Mol Cell Proteomics 7(2):326–346

    Article  CAS  PubMed  Google Scholar 

  10. Hubner NC et al (2010) Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol 189(4):739–754

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Blagoev B et al (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21(3):315–318

    Article  CAS  PubMed  Google Scholar 

  12. Mathias RA et al (2014) Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity. Cell 159(7):1615–1625

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Miteva YV, Budayeva HG, Cristea IM (2013) Proteomics-based methods for discovery, quantification, and validation of protein-protein interactions. Anal Chem 85(2):749–768

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Nesvizhskii AI (2012) Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments. Proteomics 12(10):1639–1655

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Cristea IM et al (2006) Tracking and elucidating alphavirus-host protein interactions. J Biol Chem 281(40):30269–30278

    Article  CAS  PubMed  Google Scholar 

  16. Collins BC et al (2013) Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system. Nat Methods 10(12):1246–1253

    Article  CAS  PubMed  Google Scholar 

  17. Guise AJ et al (2012) Aurora B-dependent regulation of class IIa histone deacetylases by mitotic nuclear localization signal phosphorylation. Mol Cell Proteomics 11(11):1220–1229

    Article  PubMed Central  PubMed  Google Scholar 

  18. Oda Y et al (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci U S A 96(12):6591–6596

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Ong SE, Kratchmarova I, Mann M (2003) Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J Proteome Res 2(2):173–181

    Article  CAS  PubMed  Google Scholar 

  20. Ranish JA et al (2003) The study of macromolecular complexes by quantitative proteomics. Nat Genet 33(3):349–355

    Article  CAS  PubMed  Google Scholar 

  21. Sardiu ME et al (2008) Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci U S A 105(5):1454–1459

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Tate S et al (2013) Label-free quantitative proteomics trends for protein-protein interactions. J Proteomics 81:91–101

    Article  CAS  PubMed  Google Scholar 

  23. Choi H et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8(1):70–73

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Mellacheruvu D et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10(8):730–736

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Choi H et al (2012) Analyzing protein-protein interactions from affinity purification-mass spectrometry data with SAINT. Curr Protoc Bioinformatics. Chapter 8, Unit8.15

    Google Scholar 

  26. Diner BA et al (2015) The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA. Mol Syst Biol 11(2):787

    Article  PubMed Central  PubMed  Google Scholar 

  27. Tackett AJ et al (2005) I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res 4(5):1752–1756

    Article  CAS  PubMed  Google Scholar 

  28. Byrum SD et al (2012) ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus. Cell Rep 2(1):198–205

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Bottermann K et al (2013) Systematic analysis reveals elongation factor 2 and alpha-enolase as novel interaction partners of AKT2. PLoS One 8(6), e66045

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Di Virgilio M et al (2013) Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching. Science 339(6120):711–715

    Article  PubMed  Google Scholar 

  31. Ramanagoudr-Bhojappa R et al (2013) Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein. Nucleic Acids Res 41(2):1029–1046

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Tsai YC et al (2012) Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Mol Cell Proteomics 11(5):60–76

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Moorman NJ et al (2010) A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation. Mol Cell Proteomics 9(5):851–860

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Goldberg AD et al (2010) Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140(5):678–691

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Carabetta VJ, Silhavy TJ, Cristea IM (2010) The response regulator SprE (RssB) is required for maintaining poly(A) polymerase I-degradosome association during stationary phase. J Bacteriol 192(14):3713–3721

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Niepel M et al (2013) The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome. Mol Biol Cell 24(24):3920–3938

    Article  PubMed Central  PubMed  Google Scholar 

  37. Mann JM et al (2013) Complex formation and processing of the minor transformation pilins of Bacillus subtilis. Mol Microbiol 90(6):1201–1215

    Article  CAS  PubMed  Google Scholar 

  38. Castellana M et al (2014) Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat Biotechnol 32(10):1011–1018

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Miteva YV, Cristea IM (2014) A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity. Mol Cell Proteomics 13(1):168–183

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Conlon FL et al (2012) Immunoisolation of protein complexes from Xenopus. Methods Mol Biol 917:369–390

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Manza LL et al (2005) Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5(7):1742–1745

    Article  CAS  PubMed  Google Scholar 

  42. Wisniewski JR et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362

    Article  CAS  PubMed  Google Scholar 

  43. Erde J, Loo RR, Loo JA (2014) Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J Proteome Res 13(4):1885–1895

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Chen EI et al (2007) Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J Proteome Res 6(7):2529–2538

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Chatr-Aryamontri A et al (2015) The BioGRID interaction database: 2015 update. Nucleic Acids Res 43(database issue):D470–D478

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful for funding from NIH grants R01GM114141, R21AI102187, and R21 HD073044, an NJCCR postdoctoral fellowship to TMG, and a NSF graduate research fellowship to AJG.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, 210 Lewis Thomas Laboratory, Washington Road, Princeton, NJ, 08544, USA

    Todd M. Greco, Amanda J. Guise & Ileana M. Cristea

Authors
  1. Todd M. Greco
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amanda J. Guise
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ileana M. Cristea
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ileana M. Cristea .

Editor information

Editors and Affiliations

  1. NIDDK, National Institutes of Health, BETHESDA, Maryland, USA

    Salvatore Sechi

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Greco, T.M., Guise, A.J., Cristea, I.M. (2016). Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy. In: Sechi, S. (eds) Quantitative Proteomics by Mass Spectrometry. Methods in Molecular Biology, vol 1410. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3524-6_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3524-6_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3522-2

  • Online ISBN: 978-1-4939-3524-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation