Advertisement

An Efficient Fluorescent Protein-Based Multifunctional Affinity Purification Approach in Mammalian Cells

  • Hanhui Ma
  • Janel R. McLean
  • Kathleen L. Gould
  • Dannel McCollumEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

Knowledge of an individual protein’s modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501–511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

Key words

Multifunctional affinity purification (MAP) TAP Fluorescent protein Venus FACS Protein complex MudPIT LC-MS/MS Proteomics 

Notes

Acknowledgements

This work was supported by NIH grant GM068786 to D.M., NCI T32CA119925 to J.R.M., and Howard Hughes Medical Institute for K.L.G.

References

  1. 1.
    Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032PubMedCrossRefGoogle Scholar
  2. 2.
    Burckstummer T, Bennett KL, Preradovic A, Schutze G, Hantschel O, Superti-Furga G, Bauch A (2006) An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3:1013–1019PubMedCrossRefGoogle Scholar
  3. 3.
    Tsai A, Carstens RP (2006) An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1:2820–2827PubMedCrossRefGoogle Scholar
  4. 4.
    Gloeckner CJ, Boldt K, Schumacher A, Roepman R, Ueffing M (2007) A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 7:4228–4234PubMedCrossRefGoogle Scholar
  5. 5.
    Glatter T, Wepf A, Aebersold R, Gstaiger M (2009) An integrated workflow for charting the human interaction proteome: insights into the PP2A system. Mol Syst Biol 5:237PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Cheeseman IM, Desai A (2005) A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci STKE 2005(266):pl1PubMedGoogle Scholar
  7. 7.
    Kobayashi T, Morone N, Kashiyama T, Oyamada H, Kurebayashi N, Murayama T (2008) Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS One 3:e3822PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Ma H, McLean JR, Chao LF, Mana-Capelli S, Paramasivam M, Hagstrom KA, Gould KL, McCollum D (2012) A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms. Mol Cell Proteomics 11:501–511PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Zhang F, Wang J, Xu J, Zhang Z, Koppetsch BS, Schultz N, Vreven T, Meignin C, Davis I, Zamore PD, Weng Z, Theurkauf WE (2012) UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery. Cell 151:871–884Google Scholar
  10. 10.
    Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918PubMedCrossRefGoogle Scholar
  11. 11.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909PubMedCrossRefGoogle Scholar
  12. 12.
    Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B, Baldi P, Huang L, Kaiser P (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. Mol Cell Proteomics 5:737–748PubMedCrossRefGoogle Scholar
  13. 13.
    Ota K, Kito K, Iemura S, Natsume T, Ito T (2008) A parallel affinity purification method for selective isolation of polyubiquitinated proteins. Proteomics 8:3004–3007PubMedCrossRefGoogle Scholar
  14. 14.
    McDonald W, Ohi R, Miyamoto D, Mitchison T, Yates JR III (2002) Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int J Mass Spectr 219:245–251CrossRefGoogle Scholar
  15. 15.
    Roberts-Galbraith RH, Chen JS, Wang J, Gould KL (2009) The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring. J Cell Biol 184:113–127PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Ma ZQ, Tabb DL, Burden J, Chambers MC, Cox MB, Cantrell MJ, Ham AJ, Litton MD, Oreto MR, Schultz WC, Sobecki SM, Tsui TY, Wernke GR, Liebler DC (2011) Supporting tool suite for production proteomics. Bioinformatics 27:3214–3215PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Tabb DL, Fernando CG, Chambers MC (2007) MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis. J Proteome Res 6:654–661PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Zhang B, Chambers MC, Tabb DL (2007) Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J Proteome Res 6:3549–3557PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Ma ZQ, Dasari S, Chambers MC, Litton MD, Sobecki SM, Zimmerman LJ, Halvey PJ, Schilling B, Drake PM, Gibson BW, Tabb DL (2009) IDPicker 2.0: improved protein assembly with high discrimination peptide identification filtering. J Proteome Res 8:3872–3881PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T (2011) Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27:431–432PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Hanhui Ma
    • 1
  • Janel R. McLean
    • 2
  • Kathleen L. Gould
    • 2
  • Dannel McCollum
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular PharmacologyUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Department of Cell and Developmental BiologyVanderbilt University School of MedicineNashvilleUSA

Personalised recommendations