Advertisement

Journal of Structural and Functional Genomics

, Volume 6, Issue 2–3, pp 129–134 | Cite as

High-throughput Limited Proteolysis/Mass Spectrometry for Protein Domain Elucidation

  • Xia Gao
  • Kevin Bain
  • Jeffery B. Bonanno
  • Michelle Buchanan
  • Davin Henderson
  • Don Lorimer
  • Curtis Marsh
  • Julie A. Reynes
  • J. Michael Sauder
  • Ken Schwinn
  • Chau Thai
  • Stephen K. Burley
Article

Abstract

High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1–7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (http://www.nigms.nih.gov/psi). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.

Key words

automation domain definition limited proteolysis LPMS mass spectrometry protein domain 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Cabral, J.H.M., Lee, A., Cohen, S.L., Chait, B.T., Li, M., Mackinnon, R. 1998Cell95649655CrossRefPubMedGoogle Scholar
  2. 2.
    Cohen, S.L., Ferré-D’Amaré, A.R., Burley, S.K., Chait, B.T. 1995Protein Sci.410881099PubMedGoogle Scholar
  3. 3.
    Cohen, S.L., Chait, B. 2001Annu. Rev. Biophys. Biomol. Struct.306785CrossRefPubMedGoogle Scholar
  4. 4.
    Marcotrigiano, J., Gingras, A.C., Sonenberg, N., Burley, S.K. 1997Cell89951961CrossRefPubMedGoogle Scholar
  5. 5.
    Lee, C.H., Saksela, K., Mirza, U.A., Chait, B.T., Kuriyan, J. 1996Cell85931942CrossRefPubMedGoogle Scholar
  6. 6.
    Xie, X., Kokubo, T., Cohen, S.L., Mirza, U.A., Hoffmann, A., Chait, B.T., Roeder, R.G., Nakatani, Y., Burley, S.K. 1996Nature28316322CrossRefGoogle Scholar
  7. 7.
    Zhang, G., Liu, Y., Qin, J., Vo, B., Tang, W., Ruoho, A.E., Hurley, J.H. 1997Protein Sci.6903908PubMedGoogle Scholar
  8. 8.
    Cadene, M., Chait, B. 2000Anal. Chem.2256555658CrossRefGoogle Scholar
  9. 9.
    Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E.L., Studholme, D.J., Yeats, C., Eddy, S.R. 2004Nucleic Acids Res.32D138141CrossRefPubMedGoogle Scholar
  10. 10.
    Jones, D.T. 1999J. Mol. Biol.292195202CrossRefPubMedGoogle Scholar
  11. 11.
    Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J. 1997Nucleic Acids Res.2533893402CrossRefPubMedGoogle Scholar
  12. 12.
    Kim, K.M., Yi, E.C., Baker, D., Zhang, K.Y.J. 2001Acta Crystallogr.D57759762Google Scholar
  13. 13.
    Bucher, M.H., Evdokimov, A.G., Waugh, D.S. 2002Acta Crystallogr.D58392397Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Xia Gao
    • 1
  • Kevin Bain
    • 1
  • Jeffery B. Bonanno
    • 1
  • Michelle Buchanan
    • 1
  • Davin Henderson
    • 1
  • Don Lorimer
    • 1
  • Curtis Marsh
    • 1
  • Julie A. Reynes
    • 1
  • J. Michael Sauder
    • 1
  • Ken Schwinn
    • 1
  • Chau Thai
    • 1
  • Stephen K. Burley
    • 1
  1. 1.Structural GenomiX, IncSan DiegoUSA

Personalised recommendations